MMSC1 - an MMAC1 interacting protein

ABSTRACT

The present invention is directed to the MMSC1 gene, its protein product and the use of the protein to (i) detect mutant MMAC1 proteins, (ii) screen for drugs which can be used for suppressing tumor growth and (iii) identify proteins which interact with the MMAC1 gene or are involved in the tumor suppression pathway of the MMAC1 gene.

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application is a divisional application of U.S.patent application Ser. No. 09/233,086, filed Jan. 19,1999. The presentapplication is related to U.S. provisional application 60/071,861, filedJan. 20,1998, incorporated herein by reference, and claims prioritythereto under 35 U.S.C. § 119(e).

BACKGROUND OF THE INVENTION

[0002] The present invention is directed to the MMSC1 gene, its proteinproduct and the use of the protein to (i) detect mutant MMAC1 proteins,(ii) screen for drugs which can be used for suppressing tumor growth and(iii) identify proteins which interact with the MMAC1 gene or areinvolved in the tumor suppression pathway of the MMAC1 gene.

[0003] The publications and other materials used herein to illuminatethe background of the invention or provide additional details respectingthe practice, are incorporated by reference, and for convenience arerespectively grouped in the appended List of References.

[0004] A number of genetic alterations are involved in the oncogenesisof glioblastoma multiforme, including inactivation of p53, p16, RB,amplification of the gene encoding epidermal growth factor receptor andseveral other molecular alterations (Louis & Gusella, 1995). However themost common genetic alteration is the deletion of large regions or anentire copy of chromosome 10 (Fults et al., 1990; Rahseed et al., 1992).Recently, the tumor suppressor gene MMAC1 (Steck et al., 1997), alsoknown as PTEN (Li et al., 1997) or TEP1 (Li & Sun, 1997) was mapped to10q23 and shown to be mutated in 17-24% of xenografted and primaryglioblastomas, 14% of breast cancer samples and 25% of kidney carcinomas(Steck et al., 1997). The mutation frequency in established cell linesof these tumor types is somewhat higher. In addition to this predictedinvolvement in sporadic cancer, germ-line MMAC1 mutations have beendetected in two autosomal dominant disorders, Cowden disease (Nelen etal., 1997; Liaw et al., 1997), a syndrome that confers an elevated riskfor tumors of breast, thyroid and skin, and Bannayan-Zonana syndrome(Marsh et al., 1997), a condition characterized by macrocephaly,lipomas, intestinal hamartomatous polyps, vascular malformations andsome skin disorders. Mutations of MMAC1 in primary endometrialcarcinomas (Kong et al., 1997) and in juvenile polyposis coli (Olschwanget al., 1998) have also been seen.

[0005] The predicted protein product of the MMAC1 gene has severalregions of homology with other proteins. The MMAC1 protein has an animoterminal domain with extensive homology to tensin, a protein thatinteracts with actin filaments at focal adhesions, and with auxilin, aprotein involved in synaptic vesicle transport. The MMAC1 protein alsohas a region with extensive homology to protein tyrosine phosphatases(Steck et al., 1997; Li et al., 1997). Mutations of MMAC1 in tumors, itscytoplasmic localization (Li & Sun, 1997) and its intrinsic phosphataseactivity (Li & Sun, 1997; Myers et al., 1997) suggested that itsactivity could be important in some aspect of tumor progression,possibly to counteract the oncogenic effect of a specific proteintyrosine kinase. In addition, MMAC1 is rapidly down-regulated by TGF, incells sensitive to its cell growth and cell adhesion regulatoryproperties (Li & Sun, 1997).

[0006] Experiments on glioma cell growth have shown that MMAC1 is aprotein phosphatase that exhibits functional and specificgrowth-suppressing activity. In such experiments, the introduction ofHA-tagged MMAC1 into glioma cells containing endogenous mutant allelescaused growth suppression, but was without effect in cells containingHA-tagged MMAC1 (Fumari et al., 1997). The ectopic expression of MMAC1alleles, which carried mutations found in primary tumors and have beenshown or are expected to inactivate its phosphatase activity, causedlittle growth suppression (Furnari et al., 1997). Although theseactivities of MMAC1 are known, the mechanisms of tumor suppression byMMAC1 and the interaction of the MMAC1 protein with other proteins arenot well understood.

[0007] Many cytosolic signaling proteins and cytoskeletal proteins arecomposed of modular units of small protein-protein interactive domainsthat allow reversible and regulated assembly into larger proteincomplexes. These domains include the Src-homology SH2 and SH3 domains(Schlessinger, 1994; Pawson, 1994), pleckstrin-homology (PH) domains(Lemmon et al., 1996; Shaw, 1996), phosphotyrosine-binding (PTB) domains(Harrison, 1996; van der Greer & Pawson, 1995; Kavanaugh et al., 1995)and postsynaptic density protein, disc-large, zo-1 (PDZ) domains (Woods& Bryant, 1991; Dho et al., 1992; Woods & Bryant, 1993; Kennedy, 1995;Kornau et al., 1995). So far, PDZ domains have been found in more than50 proteins (Tsunoda et al., 1997), and many proteins have multiple PDZdomains (Pawson & Scott, 1997). For a review of PDZ domains, as well asthe other protein-protein interactive domains, see Pawson & Scott(1997).

[0008] A distinguishing feature of PDZ domains is their recognition ofshort peptides at the carboxyl terminal end of proteins. For example,one family of PDZ domains selected peptides with the consensus motifGlu-(Ser/Thr)-Xaa-(Val/Ile) (SEQ ID NO: 1) at the carboxy terminus,whereas a second family of PDZ domains selected peptides withhydrophobic or aromatic side chains at the carboxy terminal threeresidues (Songyang et al., 1997). The presence of multiple PDZ domainsin proteins may have at least two important consequences. An individualPDZ-containing protein could bind several subunits of a particularchannel thereby inducing channel aggregations. Furthermore, theindividual domains of a protein can have distinct binding specificitiesthereby inducing the formation of clusters that contain heterogeneousgroups of proteins.

[0009] One example of this latter consequence of multiple PDZ domains isthe InaD protein which contains five PDZ domains and acts as ascaffolding protein to organize the light-activated signaling events inDrosophila (Shieh & Zhu, 1996; Tsunoda et al., 1997). InaD associatesthrough distinct PDZ domains with a calcium channel(TRP), phospholipaseC-β (the target of rhodopsin-activated heterotrimeric guaninenucleotide-binding protein (Gqα)) and protein kinase C.

[0010] Two further properties of PDZ domains or proteins which containthem may expand their potential activities. First, some PDZ domains maybind internal peptide sequences and, indeed, have a propensity toundergo homotypic or heterotypic interactions with other PDZ domains(Brenman et al., 1996). Second, proteins with PDZ domains frequentlycontain other interaction modules, including SH3 and LIM domains, andcatalytic elements such a tyrosine phosphatase or nitric oxide synthasedomains. PDZ domains may therefore both coordinate the localization andclustering of receptors and channels, and provide a bridge to thecytoskeleton or intracellular signaling pathways.

[0011] It is desired to determine the mechanisms of tumor suppressionfor MMAC1 and to identify proteins which interact with the MMAC1protein. Such proteins can be used to assay for mutated MMAC1 proteinsand/or screen potential drugs for suppressing tumor growth and/oridentify additional proteins which interact with MMAC1.

SUMMARY OF THE INVENTION

[0012] The present invention is directed to the MMSC1 gene, its proteinproduct and the use of the protein to (i) detect mutant MMAC1 proteins,(ii) screen for drugs which can be used for suppressing tumor growth and(iii) identify proteins which interact with the MMAC1 gene or areinvolved in the tumor suppression pathway of the MMAC1 gene.

[0013] Using yeast two-hybrid screening, it has been found MMAC1 bindsto a protein herein named MMSC1. The nucleotide sequence is set forth asSEQ ID NO: 2, and the amino acid sequence is set forth as SEQ ID NO: 3.It has been found MMSC1 has 11 PDZ domains and that one or more of thesedomains interacts specifically with the three carboxyl terminal aminoacids of MMAC1. Specifically, it has been found that PDZ domain number 7interacts with MMAC1. Since MMSC1 contains 11 PDZ domains and interactswith MMAC1, a known tumor suppressor having a region of homology withprotein tyrosine phosphatases, MMSC1 acts as a scaffolding protein in acommon biochemical pathway with MMAC1. These characteristics indicatethat the interaction between MMAC1 and MMSC1 is required for the tumorsuppressor activity of MMAC1.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1 shows a diagram of MMSC1 indicating the position of the 11PDZ domains and the overlap of the two mouse cDNA clones.

[0015]FIG. 2 shows an alignment of the first 300 nucleotides of humanMMSC1 (H.s._MMSC1) with its translation product (H.s._MMSC1.pep) and thecorresponding sequence from the mouse ortholog (M.m._MMSC1; SEQ ID NO:4), as determined from an analysis of the sequence from the above notedclones, with its translation product (M.m._MMSC1.pep; SEQ ID NO: 5).

SUMMARY OF SEQUENCE LISTING

[0016] SEQ ID NO: 1 is a consensus motif to which one family of PDZdomains interacts. SEQ ID NO: 2 is the nucleotide sequence for the MMSC1gene. SEQ ID NO: 3 is the amino acid sequence for the MMSC1 protein. SEQID NO: 4 is the nucleotide sequence for the 5′ end of a fragment of themouse homolog. SEQ ID NO: 5 is the amino acid sequence for theN-terminus fragment of a mouse homolog. SEQ ID NO: 6 is the 15C-terminal amino acids of MMAC1. SEQ ID NO: 7 is the SH3 bindingpeptide. SEQ ID NO: 8 is the AF6 binding peptide. SEQ ID NO: 9 is theMMAC1 binding peptide. SEQ ID NOs: 10-65 are primers for PCRamplification of the MMSC1 gene.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is directed to the MMSC1 gene, its proteinproduct and the use of the protein to (i) detect mutant MMAC1 proteins,(ii) screen for drugs which can be used for suppressing tumor growth and(iii) identify proteins which interact with the MMAC1 gene or areinvolved in the tumor suppression pathway of the MMAC1 gene.

[0018] Using yeast two-hybrid screening, it has been found MMAC1 bindsto a protein herein named MMSC1. The nucleotide sequence is set forth asSEQ ID NO: 2, and the amino acid sequence is set forth as SEQ ID NO: 3.It has been found MMSC1 has 11 PDZ domains and that one or more of thesedomains interacts specifically with the three carboxyl terminal aminoacids of MMAC1. Specifically, it has been found that PDZ domain number 7interacts with MMAC1. Since MMSC1 contains 11 PDZ domains and interactswith MMAC1, a known tumor suppressor having a region of homology withprotein tyrosine phosphatases, MMSC1 acts as a scaffolding protein in acommon biochemical pathway with MMAC1. These characteristics indicatethat the interaction between MMAC1 and MMSC1 is required for the tumorsuppressor activity of MMAC1.

[0019] The evidence presented herein shows that the function of MMSC1 isto make a scaffold that binds to MMAC1, the phosphatase substrate(s),and the (probably oncogene) tyrosine kinase(s). Thus, a valuable drugwill be one that can prevent binding of either the substrate(s) or thetyrosine kinases(s) to MMSC1.

[0020] The yeast two-hybrid screening assay described herein identifiedtwo clones encoding bona fide MMAC1 interacting proteins. The cloneswere identified pzdk5 and pdzk21. A search of GenBank with the sequencesof pzdk5 and pdzk21 revealed that they could be assembled with a partialcDNA sequence AJ001306, to generate the complete coding sequence of agene named MMSC1 set forth in SEQ ID NO: 2. dBEST sequences from twomouse cDNA clones (GenBank accession numbers AA030135 and W50755; IMAGEclone numbers 457904 and 356188) suggested that they might contain thestart and stop codons, respectively, of the mouse ortholog of MMSC1.Sequencing of these clones revealed that this was indeed the case andconfirmed the assignment of the translation start and stop codons inMMSC1.

[0021] As previously noted, SEQ ID NO: 2 sets forth the nucleotidesequence for MMSC1. However, it has been found that the mRNA for MMSC1is subject to alternate splicing. On the basis of the sequence forMMSC1, genomic clones have been isolated and are being analyzed todetermine splice junctions and alternate splicing for the mRNA. Inaddition, the PDZ domains of MMSC1 are analyzed in the yeast two-hybridassay to identify other proteins which interact with MMSC1 andconsequently are involved in the MMAC1 tumor suppressor pathway.

[0022] Since MMSC1 is an MMAC1 interacting protein that is involved intumor suppression activity in the MMAC1 pathway, mutations in the MMSC1gene which affect the interaction of MMSC1 with MMAC1 or affect theinteraction of other proteins with MMAC1 as a result of the scaffoldingeffect of MMSC1 will interfere with the MMAC1 tumor suppressor pathwayand lead to tumorigenesis. Thus, an additional aspect of the presentinvention is the screening of MMSC1 for such mutations usingconventional techniques. Such methods may further comprise the step ofamplifying a portion of the MMSC1 gene, and may further include a stepof providing a set of polynucleotides which are primers foramplification of said portion of the MMSC1 gene. The method is usefulfor identifying mutations for use in either diagnosis of cancer orprognosis of cancer. Since such variants can now be detected earlier,i.e., before symptoms appear, and more definitively, better treatmentoptions will be available in those individuals identified as havingharmful mutations in MMSC1.

[0023] The present invention is directed to the determination that theMMSC1 binds to the C-terminal region of MMAC1 and is involved in acommon pathway with MMAC1 which is a known tumor suppressor. Since manyof the mutations in MMAC1 are frameshift or nonsense mutations whichconsequently alter the C-terminus of MMAC1, MMSC1 can be used to assayfor normal or mutated MMAC1 proteins using conventional techniques.

[0024] Finally, the present invention is directed to a method forscreening drug candidates to identify drugs useful for treating orpreventing cancer. Drug screening is performed by expressing mutantMMSC1 and assaying the effect of a drug candidate on the binding ofMMSC1 with MMAC1. Similarly, one can test the effect of a drug candidateon the binding of wild-type MMSC1 with a mutant MMAC1. Such assays canbe performed in vitro or in vivo, such as in oocytes, mammalian cells ortransgenic animals. Other assays may test the ability of a drug, whereinthe drug may be, e.g., a peptide, to replace the activity of MMSC1 suchthat the drug plus MMAC1 will work in concert similar to the normalwild-type interactions of MMSC1 and MMAC1. Again, similar assays may beperformed to screen for drugs which replace a mutant MMAC1 and will bindto wild-type MMSC1 to replace the MMAC1 function which is lacking as aresult of a mutated MMAC1.

[0025] According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type MMSC1 gene is detected. Inaddition, the method can be performed by detecting the wild-type MMSC1gene and confirming the lack of a cause of cancer as a result of thislocus. “Alteration of a wild-type gene” encompasses all forms ofmutations including deletions, insertions and point mutations in thecoding and noncoding regions. Deletions may be of the entire gene or ofonly a portion of the gene. Point mutations may result in stop codons,frameshift mutations or amino acid substitutions. Somatic mutations arethose which occur only in certain tissues and are not inherited in thegermline. Germline mutations can be found in any of a body's tissues andare inherited. Point mutational events may occur in regulatory regions,such as in the promoter of the gene, leading to loss or diminution ofexpression of the mRNA. Point mutations may also abolish proper RNAprocessing, leading to loss of expression of the MMSC1 gene product, orto a decrease in mRNA stability or translation efficiency.

[0026] Useful diagnostic techniques include, but are not limited tofluorescent in situ hybridization (FISH), direct DNA sequencing, PFGEanalysis, Southern blot analysis, single stranded conformation analysis(SSCA), RNase protection assay, allele-specific oligonucleotide (ASO),dot blot analysis, hybridization using nucleic acid modified with goldnanoparticles and PCR-SSCP, as discussed in detail further below. Alsouseful is the recently developed technique of DNA microchip technology.

[0027] The presence of cancer due to a germline mutation at this locusmay be ascertained by testing any tissue of a human for mutations of theMMSC1 gene. For example, a person who has inherited a germline MMSC1mutation, especially one which alters the interaction of MMSC1 withMMAC1, would be prone to develop cancer. This can be determined bytesting DNA from any tissue of the person's body. Most simply, blood canbe drawn and DNA extracted from the cells of the blood. In addition,prenatal diagnosis can be accomplished by testing fetal cells, placentalcells or amniotic cells for mutations of the MMSC1 gene. Alteration of awild-type MMSC1 allele, whether, for example, by point mutation ordeletion, can be detected by any of the means discussed herein.

[0028] There are several methods that can be used to detect DNA sequencevariation. Direct DNA sequencing, either manual sequencing or automatedfluorescent sequencing can detect sequence variation. Another approachis the single-stranded conformation polymorphism assay (SSCP) (Orita etal., 1989). This method does not detect all sequence changes, especiallyif the DNA fragment size is greater than 200 bp, but can be optimized todetect most DNA sequence variation. The reduced detection sensitivity isa disadvantage, but the increased throughput possible with SSCP makes itan attractive, viable alternative to direct sequencing for mutationdetection on a research basis. The fragments which have shifted mobilityon SSCP gels are then sequenced to determine the exact nature of the DNAsequence variation. Other approaches based on the detection ofmismatches between the two complementary DNA strands include clampeddenaturing gel electrophoresis (CDGE) (Sheffield et al., 1991),heteroduplex analysis (HA) (White et al., 1992) and chemical mismatchcleavage (CMC) (Grompe et al., 1989). None of the methods describedabove will detect large deletions, duplications or insertions, nor willthey detect a regulatory mutation which affects transcription ortranslation of the protein. Other methods which might detect theseclasses of mutations such as a protein truncation assay or theasymmetric assay, detect only specific types of mutations and would notdetect missense mutations. A review of currently available methods ofdetecting DNA sequence variation can be found in a recent review byGrompe (1993). Once a mutation is known, an allele specific detectionapproach such as allele specific oligonucleotide (ASO) hybridization canbe utilized to rapidly screen large numbers of other samples for thatsame mutation. Such a technique can utilize probes which are labeledwith gold nanoparticles to yield a visual color result (Elghanian etal., 1997).

[0029] A rapid preliminary analysis to detect polymorphisms in DNAsequences can be performed by looking at a series of Southern blots ofDNA cut with one or more restriction enzymes, preferably with a largenumber of restriction enzymes. Each blot contains a series of normalindividuals and a series of cancer cases. Southern blots displayinghybridizing fragments differing in length from control DNA when probedwith sequences near or including the MMSC1 locus indicate a possiblemutation. If restriction enzymes which produce very large restrictionfragments are used, then pulsed field gel electrophoresis (PFGE) isemployed.

[0030] Detection of point mutations may be accomplished amplification,e.g., PCR, from genomic or cDNA and sequencing the amplified nucleicacid or by molecular cloning of the MMSC1 allele and sequencing theallele using techniques well known in the art.

[0031] There are six well known methods for a more complete, yet stillindirect, test for confirming the presence of a susceptibilityallele: 1) single stranded conformation analysis (SSCP) (Orita et al.,1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell etal., 1990; Sheffield et al., 1989); 3) RNase protection assays(Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specificoligonucleotides (ASOs) (Conner et al., 1983); 5) the use of proteinswhich recognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, 1991); and 6) allele-specific PCR (Rano and Kidd, 1989). Forallele-specific PCR, primers are used which hybridize at their 3′ endsto a particular MMSC1 mutation. If the particular mutation is notpresent, an amplification product is not observed. AmplificationRefractory Mutation System (ARMS) can also be used, as disclosed inEuropean Patent Application Publication No. 0332435 and in Newton etal.,1989. Insertions and deletions of genes can also be detected bycloning, sequencing and amplification. In addition, restriction fragmentlength polymorphism (RFLP) probes for the gene or surrounding markergenes can be used to score alteration of an allele or an insertion in apolymorphic fragment. Such a method is particularly useful for screeningrelatives of an affected individual for the presence of the mutationfound in that individual. Other techniques for detecting insertions anddeletions as known in the art can be used.

[0032] In the first three methods (SSCP, DGGE and RNase protectionassay), a new electrophoretic band appears. SSCP detects a band whichmigrates differentially because the sequence change causes a differencein single-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild-typesequences.

[0033] Mismatches, according to the present invention, are hybridizednucleic acid duplexes in which the two strands are not 100%complementary. Lack of total homology may be due to deletions,insertions, inversions or substitutions. Mismatch detection can be usedto detect point mutations in the gene or in its mRNA product. Whilethese techniques are less sensitive than sequencing, they are simpler toperform on a large number of samples. An example of a mismatch cleavagetechnique is the RNase protection method. In the practice of the presentinvention, the method involves the use of a labeled riboprobe which iscomplementary to the human wild-type MMSC1 gene coding sequence. Theriboprobe and either mRNA or DNA isolated from the person are annealed(hybridized) together and subsequently digested with the enzyme RNase Awhich is able to detect some mismatches in a duplex RNA structure. If amismatch is detected by RNase A, it cleaves at the site of the mismatch.Thus, when the annealed RNA preparation is separated on anelectrophoretic gel matrix, if a mismatch has been detected and cleavedby RNase A, an RNA product will be seen which is smaller than the fulllength duplex RNA for the riboprobe and the mRNA or DNA. The riboprobeneed not be the full length of the mRNA or gene but can be a segment ofeither. If the riboprobe comprises only a segment of the mRNA or gene,it will be desirable to use a number of these probes to screen the wholemRNA sequence for mismatches.

[0034] In similar fashion, DNA probes can be used to detect mismatches,through enzymatic or chemical cleavage. See, e.g., Cotton et al., 1988;Shenk et al., 1975; Novack et al., 1986. Alternatively, mismatches canbe detected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, 1988. Witheither riboprobes or DNA probes, the cellular mRNA or DNA which mightcontain a mutation can be amplified using PCR (see below) beforehybridization. Changes in DNA of the MMSC1 gene can also be detectedusing Southern hybridization, especially if the changes are grossrearrangements, such as deletions and insertions.

[0035] DNA sequences of the MMSC1 gene which have been amplified by useof PCR may also be screened using allele-specific probes. These probesare nucleic acid oligomers, each of which contains a region of the genesequence harboring a known mutation. For example, one oligomer may beabout 30 nucleotides in length, corresponding to a portion of the genesequence. By use of a battery of such allele-specific probes, PCRamplification products can be screened to identify the presence of apreviously identified mutation in the gene. Hybridization ofallele-specific probes with amplified MMSC1 sequences can be performed,for example, on a nylon filter. Hybridization to a particular probeunder high stringency hybridization conditions indicates the presence ofthe same mutation in the tissue as in the allele-specific probe.

[0036] The newly developed technique of nucleic acid analysis viamicrochip technology is also applicable to the present invention. Inthis technique, literally thousands of distinct oligonucleotide probesare built up in an array on a silicon chip. Nucleic acid to be analyzedis fluorescently labeled and hybridized to the probes on the chip. It isalso possible to study nucleic acid-protein interactions using thesenucleic acid microchips. Using this technique one can determine thepresence of mutations or even sequence the nucleic acid being analyzedor one can measure expression levels of a gene of interest. The methodis one of parallel processing of many, even thousands, of probes at onceand can tremendously increase the rate of analysis. Several papers havebeen published which use this technique. Some of these are Hacia et al.,1996; Shoemaker et al., 1996; Chee et al., 1996; Lockhart et al., 1996;DeRisi et al., 1996; Lipshutz et al., 1995. This method has already beenused to screen people for mutations in the breast cancer gene BRCA1(Hacia et al., 1996). This new technology has been reviewed in a newsarticle in Chemical and Engineering News (Borman, 1996) and been thesubject of an editorial (Nature Genetics, 1996). Also see Fodor (1997).

[0037] The most definitive test for mutations in a candidate locus is todirectly compare genomic MMSC1 sequences from patients with those from acontrol population. Alternatively, one could sequence messenger RNAafter amplification, e.g., by PCR, thereby eliminating the necessity ofdetermining the exon structure of the candidate gene.

[0038] Mutations from patients falling outside the coding region ofMMSC1 can be detected by examining the non-coding regions, such asintrons and regulatory sequences near or within the genes. An earlyindication that mutations in noncoding regions are important may comefrom Northern blot experiments that reveal messenger RNA molecules ofabnormal size or abundance in patients as compared to controlindividuals.

[0039] Alteration of MMSC1 mRNA expression can be detected by anytechniques known in the art. These include Northern blot analysis, PCRamplification and RNase protection. Diminished mRNA expression indicatesan alteration of the wild-type gene. Alteration of wild-type genes canalso be detected by screening for alteration of wild-type MMSC1 protein.For example, monoclonal antibodies immunoreactive with MMSC1 can be usedto screen a tissue. Lack of cognate antigen would indicate a mutation.Antibodies specific for products of mutant alleles could also be used todetect mutant gene product. Such immunological assays can be done in anyconvenient formats known in the art. These include Western blots,immunohistochemical assays and ELISA assays. Any means for detecting analtered MMSC1 protein can be used to detect alteration of the wild-typeMMSC1 gene. Functional assays, such as protein binding determinations,can be used. In addition, assays can be used which detect MMSC1biochemical function. Finding a mutant MMSC1 gene product indicatesalteration of a wild-type MMSC1 gene. One such binding assay is thebinding of MMSC1 with wild-type MMAC1. Conversely, wild-type MMSC1 orthe PDZ domain interacting with MMAC1 can be used in a protein bindingassay or biochemical function assay to detect normal or mutant MMAC1proteins, where the mutant proteins are proteins lacking a wild-typeC-terminus.

[0040] A mutant MMSC1 gene or gene product or a mutant MMAC1 can also bedetected in other human body samples, such as serum, stool, urine andsputum. The same techniques discussed above for detection of mutantgenes or gene products in tissues can be applied to other body samples.By screening such body samples, a simple early diagnosis can be achievedfor cancer resulting from a mutation in the MMSC1 gene.

[0041] The primer pairs of the present invention are useful fordetermination of the nucleotide sequence of a particular MMSC1 alleleusing PCR. The pairs of single-stranded DNA primers for MMSC1 can beannealed to sequences within or surrounding the MMSC1 gene in order toprime amplifying DNA synthesis of the gene itself. A complete set ofthese primers allows synthesis of all of the nucleotides of the genecoding sequences, i.e., the exons. The set of primers preferably allowssynthesis of both intron and exon sequences. Allele-specific primers canalso be used. Such primers anneal only to particular MMSC1 mutantalleles, and thus will only amplify a product in the presence of themutant allele as a template.

[0042] In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme site sequences appended to their 5′ends. Alternatively, primers can also be prepared with 5′ phosphorylgroups which will allow for blunt end coloning of amplied sequences.Thus, all nucleotides of the primers are derived from MMSC1 sequence orsequences adjacent to MMSC1, except for the few nucleotides necessary toform a restriction enzyme site. Such enzymes and sites are well known inthe art. The primers themselves can be synthesized using techniqueswhich are well known in the art. Generally, the primers can be madeusing oligonucleotide synthesizing machines which are commerciallyavailable. Given the sequence of MMSC1, design of particular primers iswell within the skill of the art.

[0043] The nucleic acid probes provided by the present invention areuseful for a number of purposes. They can be used in Southernhybridization to genomic DNA and in the RNase protection method fordetecting point mutations already discussed above. The probes can beused to detect PCR amplification products. They may also be used todetect mismatches with the MMSC1 gene or mRNA using other techniques.

[0044] Mutations which interfere with the function of the MMSC1 geneproduct are involved in the pathogenesis of cancer. Thus, the presenceof an altered (or a mutant) MMSC1 gene which produces a protein having aloss of function, or altered function, directly increases the risk ofcancer. In order to detect a MMSC1 gene mutation, a biological sample isprepared and analyzed for a difference between the sequence of theallele being analyzed and the sequence of the wild-type allele. MutantMMSC1 alleles can be initially identified by any of the techniquesdescribed above. The mutant alleles are then sequenced to identify thespecific mutation of the particular mutant allele. Alternatively, mutantalleles can be initially identified by identifying mutant (altered)proteins, using conventional techniques. The mutant alleles are thensequenced to identify the specific mutation for each allele. Themutations, especially those which lead to an altered function of theprotein, are then used for the diagnostic and prognostic methods of thepresent invention.

[0045] The identification of the association between the MMSC1 genemutations and cancer permits the early presymptomatic screening ofindividuals to identify those at risk for developing cancer. To identifysuch individuals, MMSC1 alleles are screened for mutations eitherdirectly or after cloning the alleles. The alleles are tested for thepresence of nucleic acid sequence differences from the normal alleleusing any suitable technique, including but not limited to, one of thefollowing methods: fluorescent in situ hybridization (FISH), direct DNAsequencing, PFGE analysis, Southern blot analysis, single strandedconformation analysis (SSCP), linkage analysis, RNase protection assay,allele specific oligonucleotide (ASO), dot blot analysis and PCR-SSCPanalysis. Also useful is the recently developed technique of DNAmicrochip technology. For example, either (1) the nucleotide sequence ofboth the cloned alleles and normal MMSC1 gene or appropriate fragment(coding sequence or genomic sequence) are determined and then compared,or (2) the RNA transcripts of the MMSC1 gene or gene fragment arehybridized to single stranded whole genomic DNA from an individual to betested, and the resulting heteroduplex is treated with Ribonuclease A(RNase A) and run on a denaturing gel to detect the location of anymismatches. Two of these methods can be carried out according to thefollowing procedures.

[0046] The alleles of the MMSC1 gene in an individual to be tested arecloned using conventional techniques. For example, a blood sample isobtained from the individual. The genomic DNA isolated from the cells inthis sample is partially digested to an average fragment size ofapproximately 20 kb. Fragments in the range from 18-21 kb are isolated.The resulting fragments are ligated into an appropriate vector. Thesequences of the clones are then determined and compared to the normalMMSC1 gene.

[0047] Alternatively, polymerase chain reactions (PCRs) are performedwith primer pairs for the 5′ region or the exons of the MMSC1 gene. PCRscan also be performed with primer pairs based on any sequence of thenormal MMSC1 gene. For example, primer pairs for one of the introns canbe prepared and utilized. Finally, RT-PCR can also be performed on themRNA. The amplified products are then analyzed by single strandedconformation polymorphisms (SSCP) using conventional techniques toidentify any differences and these are then sequenced and compared tothe normal gene sequence.

[0048] Individuals can be quickly screened for common MMSC1 genevariants by amplifying the individual's DNA using suitable primer pairsand analyzing the amplified product, e.g., by dot-blot hybridizationusing allele-specific oligonucleotide probes.

[0049] The second method employs RNase A to assist in the detection ofdifferences between the normal MMSC1 gene and defective genes. Thiscomparison is performed in steps using small (˜500 bp) restrictionfragments of the MMSC1 gene as the probe. First, the MMSC1 gene isdigested with a restriction enzyme(s) that cuts the gene sequence intofragments of approximately 5 00 bp. These fragments are separated on anelectrophoresis gel, purified from the gel and cloned individually, inboth orientations, into an SP6 vector (e.g., pSP64 or pSP65). TheSP6-based plasmids containing inserts of the MMSC1 gene fragments aretranscribed in vitro using the SP6 transcription system, well known inthe art, in the presence of [α-³²P]GTP, generating radiolabeled RNAtranscripts of both strands of the gene.

[0050] Individually, these RNA transcripts are used to formheteroduplexes with the allelic DNA using conventional techniques.Mismatches that occur in the RNA:DNA heteroduplex, owing to sequencedifferences between the MMSC1 fragment and the MMSC1 allele subclonefrom the individual, result in cleavage in the RNA strand when treatedwith RNase A. Such mismatches can be the result of point mutations orsmall deletions in the individual's allele. Cleavage of the RNA strandyields two or more small RNA fragments, which run faster on thedenaturing gel than the RNA probe itself.

[0051] Any differences which are found, will identify an individual ashaving a molecular variant of the MMSC1 gene and the consequent presenceof cancer. These variants can take a number of forms. The most severeforms would be frame shift mutations or large deletions which wouldcause the gene to code for an abnormal protein or one which wouldsignificantly alter protein expression. Less severe disruptive mutationswould include small in-frame deletions and nonconservative base pairsubstitutions which would have a significant effect on the proteinproduced, such as changes to or from a cysteine residue, from a basic toan acidic amino acid or vice versa, from a hydrophobic to hydrophilicamino acid or vice versa, or other mutations which would affectsecondary or tertiary protein structure. Silent mutations or thoseresulting in conservative amino acid substitutions would not generallybe expected to disrupt protein function.

[0052] Genetic testing will enable practitioners to identify individualsat risk for cancer at, or even before, birth. Finally, this inventionchanges our understanding of the cause and treatment of cancer.

[0053] Definitions

[0054] The present invention employs the following definitions.

[0055] “Amplification of Polynucleotides” utilizes methods such as thepolymerase chain reaction (PCR), ligation amplification (or ligase chainreaction, LCR) and amplification methods based on the use of Q-betareplicase. Also useful are strand displacement amplification (SDA),thermophilic SDA, and nucleic acid sequence based amplification (3SR orNASBA). These methods are well known and widely practiced in the art.See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990(for PCR); Wu et al., 1989a (for LCR); U.S. Pat. Nos. 5,270,184 and5,455,166 and Walker et al., 1992 (for SDA); Spargo et al., 1996 (forthermophilic SDA) and U.S. Pat. No. 5,409,818, Fahy et al., 1991 andCompton, 1991 for 3SR and NASBA. Reagents and hardware for conductingPCR are commercially available. Primers useful to amplify sequences fromthe MMSC1 region are preferably complementary to, and hybridizespecifically to sequences in the MMSC1 region or in regions that flank atarget region therein. MMSC1 sequences generated by amplification may besequenced directly. Alternatively, but less desirably, the amplifiedsequence(s) may be cloned prior to sequence analysis. A method for thedirect cloning and sequence analysis of enzymatically amplified genomicsegments has been described by Scharf, 1986.

[0056] “Analyte polynucleotide” and “analyte strand” refer to a single-or double-stranded polynucleotide which is suspected of containing atarget sequence, and which may be present in a variety of types ofsamples, including biological samples.

[0057] “Antibodies.” The present invention also provides polyclonaland/or monoclonal antibodies and fragments thereof, and immunologicbinding equivalents thereof, which are capable of specifically bindingto the MMSC1 polypeptide and fragments thereof or to polynucleotidesequences from the MMSC1 region. The term “antibody” is used both torefer to a homogeneous molecular entity, or a mixture such as a serumproduct made up of a plurality of different molecular entities.Polypeptides may be prepared synthetically in a peptide synthesizer andcoupled to a carrier molecule (e.g., keyhole limpet hemocyanin) andinjected over several months into rabbits. Rabbit sera is tested forimmunoreactivity to the MMSC1 polypeptide or fragment. Monoclonalantibodies may be made by injecting mice with the protein polypeptides,fusion proteins or fragments thereof. Monoclonal antibodies will bescreened by ELISA and tested for specific immunoreactivity with MMSC1polypeptide or fragments thereof. See, Harlow and Lane, 1988. Theseantibodies will be useful in assays as well as pharmaceuticals.

[0058] Once a sufficient quantity of desired polypeptide has beenobtained, it may be used for various purposes. A typical use is theproduction of antibodies specific for binding. These antibodies may beeither polyclonal or monoclonal, and may be produced by in vitro or invivo techniques well known in the art. For production of polyclonalantibodies, an appropriate target immune system, typically mouse orrabbit, is selected. Substantially purified antigen is presented to theimmune system in a fashion determined by methods appropriate for theanimal and by other parameters well known to immunologists. Typicalsites for injection are in footpads, intramuscularly, intraperitoneally,or intradermally. Of course, other species may be substituted for mouseor rabbit. Polyclonal antibodies are then purified using techniquesknown in the art, adjusted for the desired specificity.

[0059] An immunological response is usually assayed with an immunoassay.Normally, such immunoassays involve some purification of a source ofantigen, for example, that produced by the same cells and in the samefashion as the antigen. A variety of immunoassay methods are well knownin the art. See, e.g., Harlow and Lane, 1988, or Goding, 1986.

[0060] Monoclonal antibodies with affinities of 10⁻⁸ M⁻¹ or preferably10⁻⁹ to 10⁻¹⁰ M⁻¹ or stronger will typically be made by standardprocedures as described, e.g., in Harlow and Lane, 1988 or Goding, 1986.Briefly, appropriate animals will be selected and the desiredimmunization protocol followed. After the appropriate period of time,the spleens of such animals are excised and individual spleen cellsfused, typically, to immortalized myeloma cells under appropriateselection conditions. Thereafter, the cells are clonally separated andthe supernatants of each clone tested for their production of anappropriate antibody specific for the desired region of the antigen.

[0061] Other suitable techniques involve in vitro exposure oflymphocytes to the antigenic polypeptides, or alternatively, toselection of libraries of antibodies in phage or similar vectors. SeeHuse et al., 1989. The polypeptides and antibodies of the presentinvention may be used with or without modification. Frequently,polypeptides and antibodies will be labeled by joining, eithercovalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent agents, chemiluminescent agents,magnetic particles and the like. Patents teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulinsmay be produced (see U.S. Pat. No. 4,816,567).

[0062] “Binding partner” refers to a molecule capable of binding aligand molecule with high specificity, as for example, an antigen and anantigen-specific antibody or an enzyme and its inhibitor. In general,the specific binding partners must bind with sufficient affinity toimmobilize the analyte copy/complementary strand duplex (in the case ofpolynucleotide hybridization) under the isolation conditions. Specificbinding partners are known in the art and include, for example, biotinand avidin or streptavidin, IgG and protein A, the numerous, knownreceptor-ligand couples, and complementary polynucleotide strands. Inthe case of complementary polynucleotide binding partners, the partnersare normally at least about 15 bases in length, and may be at least 40bases in length. It is well recognized by those of skill in the art thatlengths shorter than 15 (e.g., 8 bases), between 15 and 40, and greaterthan 40 bases may also be used. The polynucleotides may be composed ofDNA, RNA, or synthetic nucleotide analogs. In addition, as disclosedherein, MMAC1 and PDZ binding peptides, as well as several otherproteins, bind to or interact with MMSC1. Each of these proteins arealso considered binding partners herein.

[0063] A “biological sample” refers to a sample of tissue or fluidsuspected of containing an analyte polynucleotide or polypeptide from anindividual including, but not limited to, e.g., plasma, serum, spinalfluid, lymph fluid, the external sections of the skin, respiratory,intestinal, and genitourinary tracts, tears, saliva, blood cells,tumors, organs, tissue and samples of in vitro cell cultureconstituents.

[0064] “Encode”. A polynucleotide is said to “encode” apolypeptide if,in its native state or when manipulated by methods well known to thoseskilled in the art, it can be transcribed and/or translated to producethe mRNA for and/or the polypeptide or a fragment thereof. Theanti-sense strand is the complement of such a nucleic acid, and theencoding sequence can be deduced therefrom.

[0065] “Isolated” or “substantially pure”. An “isolated” or“substantially pure” nucleic acid (e.g., an RNA, DNA or a mixed polymer)is one which is substantially separated from other cellular componentswhich naturally accompany a native human sequence or protein, e.g.,ribosomes, polymerases, many other human genome sequences and proteins.The term embraces a nucleic acid sequence or protein which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems.

[0066] “MMSC1 Allele” refers to normal alleles of the MMSC1 locus thatinteract with MMAC1 as well as alleles of MMSC1 carrying variations thataffect the interaction with MMAC1 and that cause cancer.

[0067] “MMSC1 Locus”, “MMSC1 Gene”, “MMSC1 Nucleic Acids” or “MMSC1Polynucleotide” each refer to polynucleotides, all of which are in theMMSC1 region, that are likely to be expressed in normal tissue, certainalleles of which adversely affect the interaction with MMAC1 and resultin cancer. The MMSC1 locus is intended to include coding sequences,intervening sequences and regulatory elements controlling transcriptionand/or translation. The MMSC1 locus is intended to include all allelicvariations of the DNA sequence.

[0068] These terms, when applied to a nucleic acid, refer to a nucleicacid which encodes a human MMSC1 polypeptide, fragment, homolog orvariant, including, e.g., protein fusions or deletions. The nucleicacids of the present invention will possess a sequence which is eitherderived from, or substantially similar to a natural MMSC1-encoding geneor one having substantial homology with a natural MMSC1-encoding gene ora portion thereof.

[0069] The polynucleotide compositions of this invention include RNA,cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense andantisense strands, and may be chemically or biochemically modified ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

[0070] The present invention provides recombinant nucleic acidscomprising all or part of the MMSC1 region. The recombinant constructmay be capable of replicating autonomously in a host cell.Alternatively, the recombinant construct may become integrated into thechromosomal DNA of the host cell. Such a recombinant polynucleotidecomprises a polynucleotide of genomic, cDNA, semi-synthetic, orsynthetic origin which, by virtue of its origin or manipulation, 1) isnot associated with all or a portion of a polynucleotide with which itis associated in nature; 2) is linked to a polynucleotide other thanthat to which it is linked in nature; or 3) does not occur in nature.

[0071] Therefore, recombinant nucleic acids comprising sequencesotherwise not naturally occurring are provided by this invention.Although the wild-type sequence may be employed, it will often bealtered, e.g., by deletion, substitution or insertion. cDNA or genomiclibraries of various types may be screened as natural sources of thenucleic acids of the present invention, or such nucleic acids may beprovided by amplification of sequences resident in genomic DNA or othernatural sources, e.g., by PCR. The choice of cDNA libraries normallycorresponds to a tissue source which is abundant in mRNA for the desiredproteins. Phage libraries are normally preferred, but other types oflibraries may be used. Clones of a library are spread onto plates,transferred to a substrate for screening, denatured and probed for thepresence of desired sequences.

[0072] The DNA sequences used in this invention will usually comprise atleast about five codons (15 nucleotides), more usually at least about7-15 codons, and most preferably, at least about 35 codons. One or moreintrons may also be present. This number of nucleotides is usually aboutthe minimal length required for a successful probe that would hybridizespecifically with a MMSC1-encoding sequence. In this context, oligomersof as low as 8 nucleotides, more generally 8-17 nucleotides, can be usedfor probes, especially in connection with chip technology.

[0073] Techniques for nucleic acid manipulation are described generally,for example, in Sambrook et al., 1989 or Ausubel et al., 1992. Reagentsuseful in applying such techniques, such as restriction enzymes and thelike, are widely known in the art and commercially available from suchvendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega,U. S. Biochemicals, New England Nuclear, and a number of other sources.The recombinant nucleic acid sequences used to produce fusion proteinsof the present invention may be derived from natural or syntheticsequences. Many natural gene sequences are obtainable from various cDNAor from genomic libraries using appropriate probes. See, GenBank,National Institutes of Health.

[0074] As used herein, a “portion” of the MMSC1 locus or region orallele is defined as having a minimal size of at least about eightnucleotides, or preferably about 15 nucleotides, or more preferably atleast about 25 nucleotides, and may have a minimal size of at leastabout 40 nucleotides. This definition includes all sizes in the range of8-40 nucleotides as well as greater than 40 nucleotides.

[0075] “MMSC1 protein” or “MMSC1 polypeptide” refers to a protein orpolypeptide encoded by the MMSC1 locus, variants or fragments thereof.The term “polypeptide” refers to a polymer of amino acids and itsequivalent and does not refer to a specific length of the product; thus,peptides, oligopeptides and proteins are included within the definitionof a polypeptide. This term also does not refer to, or excludemodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations, and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),polypeptides with substituted linkages as well as other modificationsknown in the art, both naturally and non-naturally occurring.Ordinarily, such polypeptides will be at least about 50% homologous tothe native MMSC1 sequence, preferably in excess of about 90%, and morepreferably at least about 95% homologous. Also included are proteinsencoded by DNA which hybridize under high or low stringency conditions,to MMSC1-encoding nucleic acids and closely related polypeptides orproteins retrieved by antisera to the MMSC1 protein(s).

[0076] The length of polypeptide sequences compared for homology willgenerally be at least about 16 amino acids, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues.

[0077] “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For instance, a promoter is operablylinked to a coding sequence if the promoter affects its transcription orexpression.

[0078] “Probes”. Polynucleotide polymorphisms associated with MMSC1alleles which predispose to cancer are detected by hybridization with apolynucleotide probe which forms a stable hybrid with that of the targetsequence, under highly stringent to moderately stringent hybridizationand wash conditions. If it is expected that the probes will be perfectlycomplementary to the target sequence, high stringency conditions will beused. Hybridization stringency may be lessened if some mismatching isexpected, for example, if variants are expected with the result that theprobe will not be completely complementary. Conditions are chosen whichrule out nonspecific/adventitious bindings, that is, which minimizenoise. (It should be noted that throughout this disclosure, if it issimply stated that “astringent” conditions are used that is meant to beread as “high stringency” conditions are used.) Since such indicationsidentify neutral DNA polymorphisms as well as mutations, theseindications need further analysis to demonstrate detection of a MMSC1susceptibility allele.

[0079] Probes for MMSC1 alleles may be derived from the sequences of theMMSC1 region or its cDNA. The probes may be of any suitable length,which span all or a portion of the MMSC1 region, and which allowspecific hybridization to the region. If the target sequence contains asequence identical to that of the probe, the probes may be short, e.g.,in the range of about 8-30 base pairs, since the hybrid will berelatively stable under even highly stringent conditions. If some degreeof mismatch is expected with the probe, i.e., if it is suspected thatthe probe will hybridize to a variant region, a longer probe may beemployed which hybridizes to the target sequence with the requisitespecificity.

[0080] The probes will include an isolated polynucleotide attached to alabel or reporter molecule and may be used to isolate otherpolynucleotide sequences, having sequence similarity by standardmethods. For techniques for preparing and labeling probes see, e.g.,Sambrook et al., 1989 or Ausubel et al., 1992. Other similarpolynucleotides may be selected by using homologous polynucleotides.Alternatively, polynucleotides encoding these or similar polypeptidesmay be synthesized or selected by use of the redundancy in the geneticcode. Various codon substitutions may be introduced, e.g., by silentchanges (thereby producing various restriction sites) or to optimizeexpression for a particular system. Mutations may be introduced tomodify the properties of the polypeptide, perhaps to change thepolypeptide degradation or turnover rate.

[0081] Probes comprising synthetic oligonucleotides or otherpolynucleotides of the present invention may be derived from naturallyoccurring or recombinant single- or double-stranded polynucleotides, orbe chemically synthesized. Probes may also be labeled by nicktranslation, Klenow fill-in reaction, or other methods known in the art.

[0082] Portions of the polynucleotide sequence having at least abouteight nucleotides, usually at least about 15 nucleotides, and fewer thanabout 9 kb, usually fewer than about 1.0 kb, from a polynucleotidesequence encoding MMSC1 are preferred as probes. This definitiontherefore includes probes of sizes 8 nucleotides through 9000nucleotides. The probes may also be used to determine whether mRNAencoding MMSC1 is present in a cell or tissue.

[0083] “Protein modifications or fragments” are provided by the presentinvention for MMSC1 polypeptides or fragments thereof which aresubstantially homologous to primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and bio-chemicalmodifications or which incorporate unusual amino acids. Suchmodifications include, for example, acetylation, carboxylation,phosphorylation, glycosylation, ubiquitination, labeling, e.g., withradionuclides, and various enzymatic modifications, as will be readilyappreciated by those well skilled in the art. A variety of methods forlabeling polypeptides and of substituents or labels useful for suchpurposes are well known in the art, and include radioactive isotopessuch as ³²P, ligands which bind to labeled antiligands (e.g.,antibodies), fluorophores, chemiluminescent agents, enzymes, andantiligands which can serve as specific binding pair members for alabeled ligand. The choice of label depends on the sensitivity required,ease of conjugation with the primer, stability requirements, andavailable instrumentation. Methods of labeling polypeptides are wellknown in the art. See Sambrook et al., 1989 or Ausubel et al., 1992.

[0084] Besides substantially full-length polypeptides, the presentinvention provides for biologically active fragments of thepolypeptides. Significant biological activities include ligand-binding,immunological activity and other biological activities characteristic ofMMSC1 polypeptides. Immunological activities include both immunogenicfunction in a target immune system, as well as sharing of immunologicalepitopes for binding, serving as either a competitor or substituteantigen for an epitope of the MMSC1 protein. As used herein, “epitope”refers to an antigenic determinant of a polypeptide. An epitope couldcomprise three amino acids in a spatial conformation which is unique tothe epitope. Generally, an epitope consists of at least five such aminoacids, and more usually consists of at least 8-10 such amino acids.Methods of determining the spatial conformation of such amino acids areknown in the art.

[0085] For immunological purposes, tandem-repeat polypeptide segmentsmay be used as immunogens, thereby producing highly antigenic proteins.Alternatively, such polypeptides will serve as highly efficientcompetitors for specific binding. Production of antibodies specific forMMSC1 polypeptides or fragments thereof is described below.

[0086] The present invention also provides for fusion polypeptides,comprising MMSC1 polypeptides and fragments. Homologous polypeptides maybe fusions between two or more MMSC1 polypeptide sequences or betweenthe sequences of MMSC1 and a related protein. Likewise, heterologousfusions may be constructed which would exhibit a combination ofproperties or activities of the derivative proteins. For example,ligand-binding or other domains may be “swapped” between different newfusion polypeptides or fragments. Such homologous or heterologous fusionpolypeptides may display, for example, altered strength or specificityof binding. Fusion partners include immunoglobulins, bacterialβ-galactosidase, trpE, protein A, β-lactamase, alpha amylase, alcoholdehydrogenase and yeast alpha mating factor. See Godowski et al., 1988.

[0087] Fusion proteins will typically be made by either recombinantnucleic acid methods, as described below, or may be chemicallysynthesized. Techniques for the synthesis of polypeptides are described,for example, in Merrifield, 1963.

[0088] “Protein purification” refers to various methods for theisolation of the MMSC1 polypeptides from other biological material, suchas from cells transformed with recombinant nucleic acids encoding MMSC1,and are well known in the art. For example, such polypeptides may bepurified by immunoaffinity chromatography employing, e.g., theantibodies provided by the present invention. Various methods of proteinpurification are well known in the art, and include those described inDeutscher, 1990 and Scopes, 1982.

[0089] The terms “isolated”, “substantially pure”, and “substantiallyhomogeneous” are used interchangeably to describe a protein orpolypeptide which has been separated from components which accompany itin its natural state. A monomeric protein is substantially pure when atleast about 60 to 75% of a sample exhibits a single polypeptidesequence. A substantially pure protein will typically comprise about 60to 90% W/W of a protein sample, more usually about 95%, and preferablywill be over about 99% pure. Protein purity or homogeneity may beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band upon staining the gel. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art which are utilized for purification.

[0090] A MMSC1 protein is substantially free of naturally associatedcomponents when it is separated from the native contaminants whichaccompany it in its natural state. Thus, a polypeptide which ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be substantiallyfree from its naturally associated components. A protein may also berendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.

[0091] A polypeptide produced as an expression product of an isolatedand manipulated genetic sequence is an “isolated polypeptide,” as usedherein, even if expressed in a homologous cell type. Synthetically madeforms or molecules expressed by heterologous cells are inherentlyisolated molecules.

[0092] “Recombinant nucleic acid” is a nucleic acid which is notnaturally occurring, or which is made by the artificial combination oftwo otherwise separated segments of sequence. This artificialcombination is often accomplished by either chemical synthesis means, orby the artificial manipulation of isolated segments of nucleic acids,e.g., by genetic engineering techniques. Such is usually done to replacea codon with a redundant codon encoding the same or a conservative aminoacid, while typically introducing or removing a sequence recognitionsite. Alternatively, it is performed to join together nucleic acidsegments of desired functions to generate a desired combination offunctions.

[0093] “Regulatory sequences” refers to those sequences normally within100 kb of the coding region of a locus, but they may also be moredistant from the coding region, which affect the expression of the gene(including transcription of the gene, and translation, splicing,stability or the like of the messenger RNA).

[0094] “Substantial homology or similarity”. A nucleic acid or fragmentthereof is “substantially homologous” (“or substantially similar”) toanother if, when optimally aligned (with appropriate nucleotideinsertions or deletions) with the other nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 60% of the nucleotide bases, usually at least about 70%, moreusually at least about 80%, preferably at least about 90%, and morepreferably at least about 95-98% of the nucleotide bases.

[0095] Alternatively, substantial homology or (similarity) exists when anucleic acid or fragment thereof will hybridize to another nucleic acid(or a complementary strand thereof) under selective hybridizationconditions, to a strand, or to its complement. Selectivity ofhybridization exists when hybridization which is substantially moreselective than total lack of specificity occurs. Typically, selectivehybridization will occur when there is at least about 55% homology overa stretch of at least about 14 nucleotides, preferably at least about65%, more preferably at least about 75%, and most preferably at leastabout 90%. See, Kanehisa, 1984. The length of homology comparison, asdescribed, may be over longer stretches, and in certain embodiments willoften be over a stretch of at least about nine nucleotides, usually atleast about 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about32 nucleotides, and preferably at least about 36 or more nucleotides.

[0096] Nucleic acid hybridization will be affected by such conditions assalt concentration, temperature, or organic solvents, in addition to thebase composition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. The stringency conditions are dependent on thelength of the nucleic acid and the base composition of the nucleic acidand can be determined by techniques well known in the art. See, e.g.,Wetmur and Davidson, 1968.

[0097] Probe sequences may also hybridize specifically to duplex DNAunder certain conditions to form triplex or other higher order DNAcomplexes. The preparation of such probes and suitable hybridizationconditions are well known in the art.

[0098] The terms “substantial homology” or “substantial identity”, whenreferring to polypeptides, indicate that the polypeptide or protein inquestion exhibits at least about 30% identity with an entirenaturally-occurring protein or a portion thereof, usually at least about70% identity, and preferably at least about 95% identity.

[0099] “Substantially similar function” refers to the function of amodified nucleic acid or a modified protein, with reference to thewild-type MMSC1 nucleic acid or wild-type MMSC1 polypeptide. Themodified polypeptide will be substantially homologous to the wild-typeMMSC1 polypeptide and will have substantially the same function. Themodified polypeptide may have an altered amino acid sequence and/or maycontain modified amino acids. In addition to the similarity of function,the modified polypeptide may have other useful properties, such as alonger half-life. The similarity of function (activity) of the modifiedpolypeptide may be substantially the same as the activity of thewild-type MMSC1 polypeptide. Alternatively, the similarity of function(activity) of the modified polypeptide may be higher than the activityof the wild-type MMSC1 polypeptide. The modified poly-peptide issynthesized using conventional techniques, or is encoded by a modifiednucleic acid and produced using conventional techniques. The modifiednucleic acid is prepared by conventional techniques. A nucleic acid witha function substantially similar to the wild-type MMSC1 gene functionproduces the modified protein described above.

[0100] Homology, for polypeptides, is typically measured using sequenceanalysis software. See, e.g., the Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

[0101] A polypeptide “fragment,” “portion” or “segment” is a stretch ofamino acid residues of at least about five to seven contiguous aminoacids, often at least about seven to nine contiguous amino acids,typically at least about nine to 13 contiguous amino acids and, mostpreferably, at least about 20 to 30 or more contiguous amino acids.

[0102] The polypeptides of the present invention, if soluble, may becoupled to a solid-phase support, e.g., nitrocellulose, nylon, columnpacking materials (e.g., Sepharose beads), magnetic beads, glass wool,plastic, metal, polymer gels, cells, or other substrates. Such supportsmay take the form, for example, of beads, wells, dipsticks, ormembranes.

[0103] “Target region” refers to a region of the nucleic acid which isamplified and/or detected. The term “target sequence” refers to asequence with which a probe or primer will form a stable hybrid underdesired conditions.

[0104] The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, and immunology. See, e.g.,Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992;Glover, 1985; Anand, 1992; Guthrie and Fink, 1991. A general discussionof techniques and materials for human gene mapping, including mapping ofhuman chromosome 1, is provided, e.g., in White and Lalouel, 1988.

[0105] Preparation of Recombinant or Chemically Synthesized NucleicAcids, Vectors, Transformation, Host Cells

[0106] Large amounts of the polynucleotides of the present invention maybe produced by replication in a suitable host cell. Natural or syntheticpolynucleotide fragments coding for a desired fragment will beincorporated into recombinant polynucleotide constructs, usually DNAconstructs, capable of introduction into and replication in aprokaryotic or eukaryotic cell. Usually the polynucleotide constructswill be suitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to (with and withoutintegration within the genome) cultured mammalian, plant, insect orother eukaryotic cell lines. The purification of nucleic acids producedby the methods of the present invention are described, e.g., in Sambrooket al., 1989 or Ausubel et al., 1992.

[0107] The polynucleotides of the present invention may also be producedby chemical synthesis, e.g., by the phosphoramidite method described byBeaucage and Carruthers, 1981 or the triester method according toMatteucci and Caruthers, 1981, and may be performed on commercial,automated oligonucleotide synthesizers. A double-stranded fragment maybe obtained from the single-stranded product of chemical synthesiseither by synthesizing the complementary strand and annealing the strandtogether under appropriate conditions or by adding the complementarystrand using DNA polymerase with an appropriate primer sequence.

[0108] Polynucleotide constructs prepared for introduction into aprokaryotic or eukaryotic host may comprise a replication systemrecognized by the host, including the intended polynucleotide fragmentencoding the desired polypeptide, and will preferably also includetranscription and translational initiation regulatory sequences operablylinked to the polypeptide encoding segment. Expression vectors mayinclude, for example, an origin of replication or autonomouslyreplicating sequence (ARS) and expression control sequences, a promoter,an enhancer and necessary processing information sites, such asribosome-binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, and mRNA stabilizing sequences.Such vectors may be prepared by means of standard recombinant techniqueswell known in the art and discussed, for example, in Sambrook et al.,1989 or Ausubel et al., 1992.

[0109] An appropriate promoter and other necessary vector sequences willbe selected so as to be functional in the host, and may include, whenappropriate, those naturally associated with the MMSC1 gene. Examples ofworkable combinations of cell lines and expression vectors are describedin Sambrook et al., 1989 or Ausubel et al., 1992; see also, e.g.,Metzger et al., 1988. Many useful vectors are known in the art and maybe obtained from such vendors as Stratagene, New England Biolabs,Promega Biotech, and others. Promoters such as the trp, lac and phagepromoters, tRNA promoters and glycolytic enzyme promoters may be used inprokaryotic hosts. Useful yeast promoters include promoter regions formetallothionein, 3-phosphoglycerate kinase or other glycolytic enzymessuch as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymesresponsible formaltose and galactose utilization, and others. Vectorsand promoters suitable for use in yeast expression are further describedin Hitzeman et al., EP 73,675A. Appropriate non-native mammalianpromoters might include the early and late promoters from SV40 (Fiers etal., 1978) or promoters derived from murine Molony leukemia virus, mousetumor virus, avian sarcoma viruses, adenovirus II, bovine papillomavirus or polyoma. Insect promoters may be derived from baculovirus. Inaddition, the construct may be joined to an amplifiable gene (e.g.,DHFR) so that multiple copies of the gene may be made. For appropriateenhancer and other expression control sequences, see also Enhancers andEukaryotic Gene Expression, Cold Spring Harbor Press, Cold SpringHarbor, New York (1983).

[0110] While such expression vectors may replicate autonomously, theymay also replicate by being inserted into the genome of the host cell,by methods well known in the art.

[0111] Expression and cloning vectors will likely contain a selectablemarker, a gene encoding a protein necessary for survival or growth of ahost cell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that a) confer resistance to antibioticsor other toxic substances, e.g. ampicillin, neomycin, methotrexate,etc., b) complement auxotrophic deficiencies, or c) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. The choice of the proper selectablemarker will depend on the host cell, and appropriate markers fordifferent hosts are well known in the art.

[0112] The vectors containing the nucleic acids of interest can betranscribed in vitro, and the resulting RNA introduced into the hostcell by well-known methods, e.g., by injection (see, Kubo et al., 1988),or the vectors can be introduced directly into host cells by methodswell known in the art, which vary depending on the type of cellularhost, including electroporation; transfection employing calciumchloride, rubidium chloride calcium phosphate, DEAE-dextran, or othersubstances; microprojectile bombardment; lipofection; infection (wherethe vector is an infectious agent, such as a retroviral genome); andother methods. See generally, Sambrook et al., 1989 and Ausubel et al.,1992. The introduction of the polynucleotides into the host cell by anymethod known in the art, including, inter alia, those described above,will be referred to herein as “transformation.” The cells into whichhave been introduced nucleic acids described above are meant to alsoinclude the progeny of such cells.

[0113] Large quantities of the nucleic acids and polypeptides of thepresent invention may be prepared by expressing the MMSC1 nucleic acidor portions thereof in vectors or other expression vehicles incompatible prokaryotic or eukaryotic host cells. The most commonly usedprokaryotic hosts are strains of Escherichia coli, although otherprokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.

[0114] Mammalian or other eukaryotic host cells, such as those of yeast,filamentous fungi, plant, insect, or amphibian or avian species, mayalso be useful for production of the proteins of the present invention.Propagation of mammalian cells in culture is per se well known. See,Jakoby and Pastan (eds.), 1979. Examples of commonly used mammalian hostcell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells,and W138, BHK, and COS cell lines. An example of a commonly used insectcell line is SF9. However, it will be appreciated by the skilledpractitioner that other cell lines may be appropriate, e.g., to providehigher expression, desirable glycosylation patterns, or other features.

[0115] Clones are selected by using markers depending on the mode of thevector construction. The marker may be on the same or a different DNAmolecule, preferably the same DNA molecule. In prokaryotic hosts, thetransformant may be selected, e.g., by resistance to ampicillin,tetracycline or other antibiotics. Production of a particular productbased on temperature sensitivity may also serve as an appropriatemarker.

[0116] Prokaryotic or eukaryotic cells transformed with thepolynucleotides of the present invention will be useful not only for theproduction of the nucleic acids and polypeptides of the presentinvention, but also, for example, in studying the characteristics ofMMSC1 polypeptide.

[0117] The probes and primers based on the MMSC1 gene sequence disclosedherein are used to identify homologous MMSC1 gene sequences and proteinsin other species. These gene sequences and proteins are used in thediagnostic/prognostic, therapeutic and drug screening methods describedherein for the species from which they have been isolated.

[0118] Methods of Use: Drug Screening

[0119] This invention is particularly useful for screening compounds byusing the MMSC1 polypeptide or binding fragment thereof in any of avariety of drug screening techniques. Since MMSC1 acts as a scaffoldthat binds to MMAC1, the phosphatase substrate(s) and the (probablyoncogene) tyrosine kinase(s), a valuable drug candidate will be a drugthat can prevent binding of either the substrate(s) or the tyrosinekinase(s) to MMSC1.

[0120] The MMSC1 polypeptide or fragment employed in such a test mayeither be free in solution, affixed to a solid support, or borne on acell surface. One method of drug screening utilizes eukaryotic orprocaryotic host cells which are stably transformed with recombinantpolynucleotides expressing the polypeptide or fragment, preferably incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. One may measure, for example,for the formation of complexes between a MMSC1 polypeptide or fragmentand the agent being tested, or examine the degree to which the formationof a complex between a MMSC1 polypeptide or fragment and a known ligand,e.g., MMAC1, is aided or interfered with by the agent being tested.

[0121] Thus, the present invention provides methods of screening fordrugs comprising contacting such an agent with a MMSC1 polypeptide orfragment thereof and assaying (i) for the presence of a complex betweenthe agent and the MMSC1 polypeptide or fragment, or (ii) for thepresence of a complex between the MMSC1 polypeptide or fragment and aligand, by methods well known in the art. In such competitive bindingassays the MMSC1 polypeptide or fragment is typically labeled. FreeMMSC1 polypeptide or fragment is separated from that present in aprotein:protein complex, and the amount of free (i.e., uncomplexed)label is a measure of the binding of the agent being tested to MMSC1 orits interference with or promotion of MMSC1 :ligand binding,respectively. One may also measure the amount of bound, rather thanfree, MMSC1. It is also possible to label the ligand rather than theMMSC1 and to measure the amount of ligand binding to MMSC1 in thepresence and in the absence of the drug being tested.

[0122] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to the MMSC1polypeptides and is described in detail in Geysen, PCT publishedapplication WO 84/03564, published on Sep. 13, 1984. Briefly stated,large numbers of different small peptide test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thepeptide test compounds are reacted with MMSC1 polypeptide and washed.Bound MMSC1 polypeptide is then detected by methods well known in theart.

[0123] Purified MMSC1 can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to capture antibodies toimmobilize the MMSC1 polypeptide on the solid phase.

[0124] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable ofspecifically binding the MMSC1 polypeptide compete with a test compoundfor binding to the MMSC1 polypeptide or fragments thereof. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants of the MMSC1polypeptide.

[0125] The above screening methods are not limited to assays employingonly MMSC1 but are also applicable to studying MMSC1-protein complexes,e.g., the complex which occurs between MMSC1 and MMAC1. The effect ofdrugs on the activity of this complex, especially when either the MMSC1or the MMSC1 binding protein (e.g., MMAC1) contains a mutation, isanalyzed.

[0126] In accordance with these methods, the following assays areexamples of assays which can be used for screening for drug candidates.

[0127] A mutant MMSC1 (per se or as part of a fusion protein) iscombined with a wild-type protein (per se or as part of a fusionprotein) to which wild-type MMSC1 binds. This combining is performed inboth the presence of a drug and the absence of the drug, and the amountof binding of the mutant MMSC1 with the wild-type protein is measured.If the amount of the binding is more in the presence of said drug thanin the absence of said drug, the drug is a drug candidate for treatingcancer resulting from a mutation in MMSC1. This assay is useful wherethe wild-type protein is a tumor suppressor, such as MMAC1.

[0128] A wild-type MMSC1 (per se or as part of a fusion protein) iscombined with a wild-type protein (per se or as part of a fusionprotein) to which wild-type MMSC1 binds. This combining is performed inboth the presence of a drug and the absence of the drug, and the amountof binding of the wild-type MMSC1 with the wild-type protein ismeasured. If the amount of the binding is more in the presence of saiddrug than in the absence of said drug, the drug is a drug candidate fortreating cancer resulting from a mutation in MMSC1. This assay is usefulwhere the wild-type protein is a tumor suppressor, such as MMAC1.

[0129] A mutant MMSC1 (per se or as part of a fusion protein) iscombined with a wild-type protein (per se or as part of a fusionprotein) to which wild-type MMSC1 binds. This combining is performed inboth the presence of a drug and the absence of the drug, and the amountof binding of the mutant MMSC1 with the wild-type protein is measured.If the amount of the binding is less in the presence of said drug thanin the absence of said drug, the drug is a drug candidate for treatingcancer resulting from a mutation in MMSC1. This assay is useful if theprotein is an oncoprotein or a substrate of the oncoprotein.

[0130] A wild-type MMSC1 (per se or as part of a fusion protein) iscombined with a wild-type protein (per se or as part of a fusionprotein) to which wild-type MMSC1 binds. This combining is performed inboth the presence of a drug and the absence of the drug, and the amountof binding of the wild-type MMSC1 with the wild-type protein ismeasured. If the amount of the binding is less in the presence of saiddrug than in the absence of said drug, the drug is a drug candidate fortreating cancer resulting from a mutation in MMSC1 or a cancer resultingfrom a mutation in MMAC1. This assay is useful if the protein is anoncoprotein or a substrate of the oncoprotein.

[0131] A mutant protein, which as a wild-type protein binds to MMSC1(per se or as part of a fusion protein) is combined with a wild-typeMMSC1 (per se or as part of a fusion protein). This combining isperformed in both the presence of a drug and the absence of the drug,and the amount of binding of the mutant protein with the wild-type MMSC1is measured. If the amount of the binding is less in the presence ofsaid drug than in the absence of said drug, the drug is a drug candidatefor treating cancer resulting from a mutation in the gene encoding theprotein.

[0132] The polypeptide of the invention may also be used for screeningcompounds developed as a result of combinatorial library technology.Combinatorial library technology provides an efficient way of testing apotential vast number of different substances for ability to modulateactivity of a polypeptide. Such libraries and their use are known in theart. The use of peptide libraries is preferred. See, for example, WO97/02048.

[0133] Briefly, a method of screening for a substance which modulatesactivity of a polypeptide may include contacting one or more testsubstances with the polypeptide in a suitable reaction medium, testingthe activity of the treated polypeptide and comparing that activity withthe activity of the polypeptide in comparable reaction medium untreatedwith the test substance or substances. A difference in activity betweenthe treated and untreated polypeptides is indicative of a modulatingeffect of the relevant test substance or substances.

[0134] Prior to or as well as being screened for modulation of activity,test substances may be screened for ability to interact with thepolypeptide, e.g., in a yeast two-hybrid system (e.g., Bartel et al.,1993). This system may be used as a coarse screen prior to testing asubstance for actual ability to modulate activity of the polypeptide.Alternatively, the screen could be used to screen test substances forbinding to a MMSC1 specific binding partner, such as MMAC1, or to findmimetics of the MMSC1 polypeptide.

[0135] Following identification of a substance which modulates oraffects polypeptide activity, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.,manufacture or formulation, or a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

[0136] Thus, the present invention extends in various aspects not onlyto a substance identified using a nucleic acid molecule as a modulatorof polypeptide activity, in accordance with what is disclosed herein,but also a pharmaceutical composition, medicament, drug or othercomposition comprising such a substance, a method comprisingadministration of such a composition comprising such a substance, amethod comprising administration of such a composition to a patient,e.g., for treatment (which may include preventative treatment) ofcancer, use of such a substance in the manufacture of a composition foradministration, e.g., for treatment of cancer, and a method of making apharmaceutical composition comprising admixing such a substance with apharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

[0137] A substance identified using as a modulator of polypeptidefunction may be peptide or non-peptide in nature. Non-peptide smallmolecules are often preferred for many in vivo pharmaceutical uses.Accordingly, a mimetic or mimic of the substance (particularly if apeptide) may be designed for pharmaceutical use.

[0138] The designing of mimetics to a known pharmaceutically activecompound is a known approach to the development of pharmaceuticals basedon a lead compound. This might be desirable where the active compound isdifficult or expensive to synthesize or where it is unsuitable for aparticular method of administration,e.g., peptides are unsuitable activeagents for oral compositions as they tend to be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis and testingis generally used to avoid randomly screening large numbers of moleculesfor a target property.

[0139] There are several steps commonly taken in the design of a mimeticfrom a compound having a given target property. First, the particularparts of the compound that are critical and/or important in determiningthe target property are determined. In the case of a peptide, this canbe done by systematically varying the amino acid residues in thepeptide, e.g., by substituting each residue in turn. Alanine scans ofpeptide are commonly used to refine such peptide motifs. These parts orresidues constituting the active region of the compound are known as itspharmacophore.

[0140] Once the pharmacophore has been found, its structure is modeledaccording to its physical properties, e.g., stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.,spectroscopic techniques, x-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modeling process.

[0141] In a variant of this approach, the three-dimensional structure ofthe ligand and its binding partner are modeled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

[0142] A template molecule is then selected onto which chemical groupswhich mimic the pharmacophore can be grafted. The template molecule andthe chemical groups grafted onto it can conveniently be selected so thatthe mimetic is easy to synthesize, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. Alternatively, where the mimetic ispeptide-based, further stability can be achieved by cyclizing thepeptide, increasing its rigidity. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimization ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

[0143] Methods of Use: Nucleic Acid Diagnosis and Diagnostic Kits

[0144] In order to detect the presence of a MMSC1 allele predisposing anindividual to cancer, a biological sample such as blood is prepared andanalyzed for the presence or absence of susceptibility alleles of MMSC1.In order to detect the presence of cancer or as a prognostic indicator,a biological sample is prepared and analyzed for the presence or absenceof mutant alleles of MMSC1. Results of these tests and interpretiveinformation are returned to the health care provider for communicationto the tested individual. Such diagnoses may be performed by diagnosticlaboratories, or, alternatively, diagnostic kits are manufactured andsold to health care providers or to private individuals forself-diagnosis.

[0145] Initially, the screening method involves amplification of therelevant MMSC1 sequences. In another preferred embodiment of theinvention, the screening method involves a non-PCR based strategy. Suchscreening methods include two-step label amplification methodologiesthat are well known in the art. Both PCR and non-PCR based screeningstrategies can detect target sequences with a high level of sensitivity.

[0146] The most popular method used today is target amplification. Here,the target nucleic acid sequence is amplified with polymerases. Oneparticularly preferred method using polymerase-driven amplification isthe polymerase chain reaction (PCR). The polymerase chain reaction andother polymerase-driven amplification assays can achieve over amillion-fold increase in copy number through the use ofpolymerase-driven amplification cycles. Once amplified, the resultingnucleic acid can be sequenced or used as a substrate for DNA probes.

[0147] When the probes are used to detect the presence of the targetsequences the biological sample to be analyzed, such as blood or serum,may be treated, if desired, to extract the nucleic acids. The samplenucleic acid may be prepared in various ways to facilitate detection ofthe target sequence, e.g., denaturation, restriction digestion,electrophoresis or dot blotting. The targeted region of the analytenucleic acid usually must be at least partially single-stranded to formhybrids with the targeting sequence of the probe. If the sequence isnaturally single-stranded, denaturation will not be required. However,if the sequence is double-stranded, the sequence will probably need tobe denatured. Denaturation can be carried out by various techniquesknown in the art.

[0148] Analyte nucleic acid and probe are incubated under conditionswhich promote stable hybrid formation of the target sequence in theprobe with the putative targeted sequence in the analyte. The region ofthe probes which is used to bind to the analyte can be made completelycomplementary to the targeted region for MMSC1. Therefore, highstringency conditions are desirable in order to prevent false positives.However, conditions of high stringency are used only if the probes arecomplementary to regions of the chromosome which are unique in thegenome. The stringency of hybridization is determined by a number offactors during hybridization and during the washing procedure, includingtemperature, ionic strength, base composition, probe length, andconcentration of formamide. These factors are outlined in, for example,Maniatis et al., 1982 and Sambrook et al., 1989. Under certaincircumstances, the formation of higher order hybrids, such as triplexes,quadraplexes, etc., may be desired to provide the means of detectingtarget sequences.

[0149] Detection, if any, of the resulting hybrid is usuallyaccomplished by the use of labeled probes. Alternatively, the probe maybe unlabeled, but may be detectable by specific binding with a ligandwhich is labeled, either directly or indirectly. Suitable labels, andmethods for labeling probes and ligands are known in the art, andinclude, for example, radioactive labels which may be incorporated byknown methods (e.g., nick translation, random priming or kinasing),biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies, goldnanoparticles and the like. Variations of this basic scheme are known inthe art, and include those variations that facilitate separation of thehybrids to be detected from extraneous materials and/or that amplify thesignal from the labeled moiety. A number of these variations arereviewed in, e.g., Matthews and Kricka, 1988; Landegren et al., 1988;U.S. Pat. No. 4,868,105; and in EPO Publication No. 225,807.

[0150] As noted above, non-PCR based screening assays are alsocontemplated in this invention. This procedure hybridizes a nucleic acidprobe (or an analog such as a methyl phosphonate backbone replacing thenormal phosphodiester), to the low level DNA target. This probe may havean enzyme covalently linked to the probe, such that the covalent linkagedoes not interfere with the specificity of the hybridization. Thisenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe enzyme conjugate and a substrate is added forenzyme detection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in a 10³-10⁶ increase insensitivity. For an example relating to the preparation ofoligodeoxynucleotide-alkaline phosphatase conjugates and their use ashybridization probes, see Jablonski et al., 1986.

[0151] Two-step label amplification methodologies are known in the art.These assays work on the principle that a small ligand (such asdigoxigenin, biotin, or the like) is attached to a nucleic acid probecapable of specifically binding MMSC1. Allele specific probes are alsocontemplated within the scope of this example and exemplary allelespecific probes include probes encompassing the predisposing mutationsof this disclosure.

[0152] In one example, the small ligand attached to the nucleic acidprobe is specifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digoxigenin is attached to the nucleic acidprobe. Hybridization is detected by an antibody-alkaline phosphataseconjugate which turns over a chemiluminescent substrate. For methods forlabeling nucleic acid probes according to this embodiment see Martin etal., 1990. In a second example, the small ligand is recognized by asecond ligand-enzyme conjugate that is capable of specificallycomplexing to the first ligand. A well known embodiment of this exampleis the biotin-avidin type of interactions. For methods for labelingnucleic acid probes and their use in biotin-avidin based assays seeRigby et al., 1977 and Nguyen et al., 1992.

[0153] It is also contemplated within the scope of this invention thatthe nucleic acid probe assays of this invention will employ a cocktailof nucleic acid probes capable of detecting MMSC1. Thus, in one exampleto detect the presence of MMSC1 in a cell sample, more than one probecomplementary to the gene is employed and in particular the number ofdifferent probes is alternatively two, three, or five different nucleicacid probe sequences. In another example, to detect the presence ofmutations in the MMSC1 gene sequence in a patient, more than one probecomplementary to these genes is employed where the cocktail includesprobes capable of binding to the allele-specific mutations identified inpopulations of patients with alterations in MMSC1. In this embodiment,any number of probes can be used, and will preferably include probescorresponding to the major gene mutations identified as predisposing anindividual to cancer.

[0154] Methods of Use: Peptide Diagnosis and Diagnostic Kits

[0155] The presence of cancer can also be detected on the basis of thealteration of wild-type MMSC1 polypeptide. Such alterations can bedetermined by sequence analysis in accordance with conventionaltechniques. More preferably, antibodies (olyclonal or monoclonal) areused to detect differences in, or the absence of MMSC1 peptides.Techniques for raising and purifying antibodies are well known in theart and any such techniques may be chosen to achieve the preparationsclaimed in this invention. In a preferred embodiment of the invention,antibodies will immunoprecipitate MMSC1 proteins from solution as wellas react with these proteins on Western or immunoblots of polyacrylamidegels. In another preferred embodiment, antibodies will detect MMSC1proteins in paraffin or frozen tissue sections, using immunocytochemicaltechniques.

[0156] Preferred embodiments relating to methods for detecting MMSC1 orits mutations include enzyme linked immunosorbent assays (ELISA),radioimmunoassays (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal and/or polyclonal antibodies. Exemplary sandwich assays aredescribed by David et al., in U.S. Patent Nos. 4,376,110 and 4,486,530,hereby incorporated by reference.

[0157] Alternatively, alterations in the MMSC1 sequence can bedetermined by detecting alterations in the interaction of MMSC1 withMMAC1 or the C-terminus of MMAC1. Wild-type MMAC1 or its C-terminus canbe bound to a solid phase and the interaction with MMSC1 assayed byconventional techniques. Analogously, alterations in MMAC1 which affectits interaction with MMS C can be detected using wild-type MMSC1 or itsPDZ domain which interacts with MMAC1 bound to a solid phase.

[0158] Methods of Use: Rational Drug Design

[0159] The goal of rational drug design is to produce structural analogsof biologically active polypeptides of interest or of small moleculeswith which they interact (e.g., agonists, antagonists, inhibitors) inorder to fashion drugs which are, for example, more active or stableforms of the polypeptide, or which, e.g., enhance or interfere with thefunction of a polypeptide in vivo. See, e.g., Hodgson, 1991. In oneapproach, one first determines the three-dimensional structure of aprotein of interest (e.g., MMSC1 polypeptide) by x-ray crystallography,by computer modeling or most typically, by a combination of approaches.Less often, useful information regarding the structure of a polypeptidemay be gained by modeling based on the structure of homologous proteins.An example of rational drug design is the development of HIV proteaseinhibitors (Erickson et al., 1990). In addition, peptides (e.g., MMSC1polypeptide) are analyzed by an alanine scan (Wells, 1991). In thistechnique, an amino acid residue is replaced by Ala, and its effect onthe peptide's activity is determined. Each of the amino acid residues ofthe peptide is analyzed in this manner to determine the importantregions of the peptide.

[0160] It is also possible to isolate a target-specific antibody,selected by a functional assay, and then to solve its crystal structure.In principle, this approach yields a pharmacore upon which subsequentdrug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced banks of peptides. Selected peptides would thenact as the pharmacore.

[0161] Thus, one may design drugs which have, e.g., improved MMSC1polypeptide activity or stability or which act as inhibitors, agonists,antagonists, etc. of MMSC1 polypeptide activity. By virtue of theavailability of cloned MMSC1 sequence, sufficient amounts of the MMSC1polypeptide may be made available to perform such analytical studies asx-ray crystallography. In addition, the knowledge of the MMSC1 proteinsequence provided herein will guide those employing computer modelingtechniques in place of, or in addition to x-ray crystallography.

[0162] Methods of Use: Gene Therapy

[0163] According to the present invention, a method is also provided ofsupplying wild-type MMSC1 function to a cell which carries a mutantMMSC1 allele. Supplying such a function should allow normal functioningof the recipient cells. The wild-type gene or a part of the gene may beintroduced into the cell in a vector such that the gene remainsextrachromosomal. In such a situation, the gene will be expressed by thecell from the extrachromosomal location. More preferred is the situationwhere the wild-type gene or a part thereof is introduced into the mutantcell in such a way that it recombines with the endogenous mutant genepresent in the cell. Such recombination requires a double recombinationevent which results in the correction of the gene mutation. Vectors forintroduction of genes both for recombination and for extrachromosomalmaintenance are known in the art, and any suitable vector may be used.Methods for introducing DNA into cells such as electroporation, calciumphosphate co-precipitation and viral transduction are known in the art,and the choice of method is within the competence of the practitioner.

[0164] As generally discussed above, the MMSC1 gene or fragment, whereapplicable, may be employed in gene therapy methods in order to increasethe amount of the expression products of such gene in cells. It may alsobe useful to increase the level of expression of the MMSC1 gene even inthose persons in which the mutant gene is expressed at a “normal” level,but the gene product is not fully functional.

[0165] Gene therapy would be carried out according to generally acceptedmethods, for example, as described by Friedman, 1991. Cells from apatient would be first analyzed by the diagnostic methods describedabove, to ascertain the production of MMSC1 polypeptide in the cells. Avirus or plasmid vector (see further details below), containing a copyof the MMSC1 gene linked to expression control elements and capable ofreplicating inside the cells, is prepared. Suitable vectors are known,such as disclosed in U.S. Pat. No. 5,252,479 and PCT publishedapplication WO 93/07282. The vector is then injected into the patient.If the transfected gene is not permanently incorporated into the genomeof each of the targeted cells, the treatment may have to be repeatedperiodically.

[0166] Gene transfer systems known in the art may be useful in thepractice of the gene therapy methods of the present invention. Theseinclude viral and nonviral transfer methods. A number of viruses havebeen used as gene transfer vectors, including papovaviruses (e.g., SV40,Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988;Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al.,1992; Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990),vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohiet al., 1990), herpes viruses including HSV and EBV (Margolskee, 1992;Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987;Freese et al., 1990), and retroviruses of avian (Brandyopadhyay andTemin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller etal., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al.,1988), and human origin (Shimada et al., 1991; Helseth et al., 1990;Page et al., 1990; Buchschacher and Panganiban, 1992). Most human genetherapy protocols have been based on disabled murine retroviruses.

[0167] Nonviral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham and van derEb, 1973; Pellicer et al., 1980); mechanical techniques, for examplemicroinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster etal., 1981; Constantini and Lacy, 1981); membrane fusion-mediatedtransfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989;Kaneda et al., 1989; Stewart et al., 1992; Nabel et al., 1990; Lim etal., 1992); and direct DNA uptake and receptor-mediated DNA transfer(Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al.,1989b; Wolffet al., 1991; Wagner et al., 1990; Wagner et al., 1991;Cotten et al., 1990; Curiel et al., 1991a; Curiel et al., 1991b).

[0168] In an approach which combines biological and physical genetransfer methods, plasmid DNA of any size is combined with apolylysine-conjugated antibody specific to the adenovirus hexon protein,and the resulting complex is bound to an adenovirus vector. Thetrimolecular complex is then used to infect cells. The adenovirus vectorpermits efficient binding, internalization, and degradation of theendosome before the coupled DNA is damaged.

[0169] Liposome/DNA complexes have been shown to be capable of mediatingdirect in vivo gene transfer. While in standard liposome preparationsthe gene transfer process is nonspecific, localized in vivo uptake andexpression have been reported in tumor deposits, for example, followingdirect in situ administration (Nabel, 1992).

[0170] Gene transfer techniques which target DNA directly to braintissue is preferred. Receptor-mediated gene transfer, for example, isaccomplished by the conjugation of DNA (usually in the form ofcovalently closed supercoiled plasmid) to a protein ligand viapolylysine. Ligands are chosen on the basis of the presence of thecorresponding ligand receptors on the cell surface of the targetcell/tissue type. These ligand-DNA conjugates can be injected directlyinto the blood if desired and are directed to the target tissue wherereceptor binding and internalization of the DNA-protein complex occurs.To overcome the problem of intracellular destruction of DNA, coinfectionwith adenovirus can be included to disrupt endosome function.

[0171] The therapy is as follows: patients who carry a MMSC1susceptibility allele are treated with a gene delivery vehicle such thatsome or all of their brain precursor cells receive at least oneadditional copy of a functional normal MMSC1 allele, respectively. Inthis step, the treated individuals have reduced risk of cancer to theextent that the effect of the susceptible allele has been countered bythe presence of the normal allele.

[0172] Methods of Use: Peptide Therapy

[0173] Peptides which have MMSC1 activity can be supplied to cells whichcarry a mutant or missing MMSC1 allele. Protein can be produced byexpression of the cDNA sequence in bacteria, for example, using knownexpression vectors. Alternatively, MMSC1 polypeptide can be extractedfrom MMSC1-producing mammalian cells. In addition, the techniques ofsynthetic chemistry can be employed to synthesize MMSC1 protein. Any ofsuch techniques can provide the preparation of the present inventionwhich comprises the MMSC1 protein. The preparation is substantially freeof other human proteins. This is most readily accomplished by synthesisin a microorganism or in vitro.

[0174] Active MMSC1 molecules can be introduced into cells bymicroinjection or by use of liposomes, for example. Alternatively, someactive molecules may be taken up by cells, actively or by diffusion.Supply of molecules with MMSC1 activity should lead to inhibition ofcancer. Other molecules with MMSC1 activity (for example, peptides,drugs or organic compounds) may also be used to effect such aninhibition. Modified polypeptides having substantially similar functionare also used for peptide therapy.

[0175] Methods of Use: Transformed Hosts

[0176] Animals for testing therapeutic agents can be selected aftermutagenesis of whole animals or after treatment of germline cells orzygotes. Such treatments include insertion of mutant MMSC1 alleles,usually from a second animal species, as well as insertion of disruptedhomologous genes. Alternatively, the endogenous MMSC1 gene of theanimals may be disrupted by insertion or deletion mutation or othergenetic alterations using conventional techniques (Capecchi, 1989;Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992;Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992;Donehower et al., 1992). After test substances have been administered tothe animals, the presence of cancer must be assessed. If the testsubstance prevents or suppresses the appearance of cancer, then the testsubstance is a candidate therapeutic agent for treatment of cancer.These animal models provide an extremely important testing vehicle forpotential therapeutic products.

[0177] Methods of Use: Transgenic/Knockout Animals and Models

[0178] In one embodiment of the invention, transgenic animals areproduced which contain a functional transgene encoding a functionalMMSC1 polypeptide or variants thereof. Transgenic animals expressingMMSC1 transgenes, recombinant cell lines derived from such animals andtransgenic embryos may be useful in methods for screening for andidentifying agents that induce or repress function of MMSC1. Transgenicanimals of the present invention also can be used as models for studyingindications such as cancers.

[0179] In one embodiment of the invention, a MMSC1 transgene isintroduced into a non-human host to produce a transgenic animalexpressing a human or murine MMSC1 gene. The transgenic animal isproduced by the integration of the transgene into the genome in a mannerthat permits the expression of the transgene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; which is incorporated herein by reference), Brinsteret al. 1985; which is incorporated herein by reference in its entirety)and in “Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition(eds., Hogan, Beddington, Costantimi and Long, Cold Spring HarborLaboratory Press, 1994; which is incorporated herein by reference in itsentirety).

[0180] It may be desirable to replace the endogenous MMSC1 by homologousrecombination between the transgene and the endogenous gene; or theendogenous gene may be eliminated by deletion as in the preparation of“knock-out” animals. Typically, a MMSC1 gene flanked by genomicsequences is transferred by microinjection into a fertilized egg. Themicroinjected eggs are implanted into a host female, and the progeny arescreened for the expression of the transgene. Transgenic animals may beproduced from the fertilized eggs from a number of animals including,but not limited to reptiles, amphibians, birds, mammals, and fish.Within a particularly preferred embodiment, transgenic mice aregenerated which overexpress MMSC1 or express a mutant form of thepolypeptide. Alternatively, the absence of a MMSC1 in “knock-out” micepermits the study of the effects that loss of MMSC1 protein has on acell in vivo. Knock-out mice also provide a model for the development ofMMSC1-related cancers.

[0181] Methods for producing knockout animals are generally described byShastry (1995, 1998) and Osterrieder and Wolf (1998). The production ofconditional knockout animals, in which the gene is active until knockedout at the desired time is generally described by Feil et al. (1996),Gagneten et al. (1997) and Lobe and Nagy (1998). Each of thesereferences is incorporated herein by reference.

[0182] As noted above, transgenic animals and cell lines derived fromsuch animals may find use in certain testing experiments. In thisregard, transgenic animals and cell lines capable of expressingwild-type or mutant MMSC1 may be exposed to test substances. These testsubstances can be screened for the ability to enhance wild-type MMSC1expression and or function or impair the expression or function ofmutant MMSC1.

[0183] Pharmaceutical Compositions and Routes of Administration

[0184] The MMSC1 polypeptides, antibodies, peptides and nucleic acids ofthe present invention can be formulated in pharmaceutical compositions,which are prepared according to conventional pharmaceutical compoundingtechniques. See, for example, Remington's Pharmaceutical Sciences, 18thEd. (1990, Mack Publishing Co., Easton, Pa.). The composition maycontain the active agent or pharmaceutically acceptable salts of theactive agent. These compositions may comprise, in addition to one of theactive substances, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known in the art. Suchmaterials should be non-toxic and should not interfere with the efficacyof the active ingredient. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,intravenous, oral, intrathecal, epineural or parenteral.

[0185] For oral administration, the compounds can be formulated intosolid or liquid preparations such as capsules, pills, tablets, lozenges,melts, powders, suspensions or emulsions. In preparing the compositionsin oral dosage form, any of the usual pharmaceutical media may beemployed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents, suspending agents, andthe like in the case of oral liquid preparations (such as, for example,suspensions, elixirs and solutions); or carriers such as starches,sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like in the case of oral solidpreparations (such as, for example, powders, capsules and tablets).Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe sugar-coated or enteric-coated by standard techniques. The activeagent can be encapsulated to make it stable to passage through thegastrointestinal tract while at the same time allowing for passageacross the blood brain barrier. See for example, WO 96/11698.

[0186] For parenteral administration, the compound may dissolved in apharmaceutical carrier and administered as either a solution of asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they may also be dissolved in cerebrospinal fluid.

[0187] The active agent is preferably administered in an therapeuticallyeffective amount. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofthe condition being treated. Prescription of treatment, e.g. decisionson dosage, timing, etc., is within the responsibility of generalpractitioners or spealists, and typically takes account of the disorderto be treated, the condition of the individual patient, the site ofdelivery, the method of administration and other factors known topractitioners. Examples of techniques and protocols can be found inRemington's Parmaceutical Sciences.

[0188] Alternatively, targeting therapies may be used to deliver theactive agent more specifically to certain types of cell, by the use oftargeting systems such as antibodies or cell specific ligands. Targetingmay be desirable for a variety of reasons, e.g. if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

[0189] Instead of administering these agents directly, they could beproduced in the target cell, e.g. in a viral vector such as describedabove or in a cell based delivery system such as described in U.S. Pat.No. 5,550,050 and published PCT application Nos. WO 92/19195, WO94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO96/40871, WO 96/40959 and WO 97/12635. designed for implantation in apatient. The vector could be targeted to the specific cells to betreated, or it could contain regulatory elements which are more tissuespecific to the target cells. The cell based delivery system is designedto be implanted in a patient's body at the desired target site andcontains a coding sequence for the active agent. Alternatively, theagent could be administered in a precursor form for conversion to theactive form by an activating agent produced in, or targeted to, thecells to be treated. See for example, EP 425,731A and WO 90/07936.

EXAMPLES

[0190] The present invention is further detailed in the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below are utilized.

Example 1 Identification of MMSC1

[0191] A yeast two-hybrid assay was performed using conventionaltechniques, such as described by Fields and Song (1989), Chevray andNathans (1992), Bartel et al. (1993) and Lee et al. (1995). Sequenceencoding the C-terminal 15 amino acids of MMAC1 (NEPFDEDQHTQITKV; SEQ IDNO: 6) plus its stop codon was generated using an oligonucleotidesynthesizer and was ligated to plasmid pGBT.C such that the codingsequence of MMAC1 was in-frame with coding sequence for the Gal4pDNA-binding domain. This plasmid construct was introduced into the yeastreporter strain J692 along with a library of activation domain fusionplasmids prepared from human kidney cDNA (Clontech). Transformants werespread onto 20-150 mm plates of yeast minimal media lacking leucine,tryptophan, and histidine, and containing 25 mM 3-amino-1,2,4-triazole(Gietz et al., 1995; Bartel and Fields, 1995). After one week incubationat 300° C., yeast colonies were assayed for expression of the lacZreporter gene by beta-galactosidase filter assay (Breeden and Naysmyth,1985). Colonies that both grew in the absence of histidine and werepositive for production of beta-galactosidase were chosen for furthercharacterization.

[0192] The activation domain plasmid was purified from positive coloniesby the smash-and-grab technique. These plasmids were introduced into E.coli DH10B (Gibco BRL) by electroporation and purified from E. coli bythe alkaline lysis method. To test for the specificity of theinteraction, specific activation domain plasmids were cotransformed intostrain J692 with plasmids encoding various DNA-binding domain fusionproteins, including fusions to C-terminal segments of MMAC1 and humanlamin C. Transformants from these experiments were assayed forexpression of the HIS3 and lacZ reporter genes. Positives that expressedreporter genes with MMAC1 constructs and not with lamin C constructsencode bona fide MMAC1-interacting proteins. These proteins wereidentified and characterized by sequence analysis of the insert of theappropriate activation domain plasmid.

[0193] Two of the clones encoding bona fide MMAC1-interacting proteinswere named pdzk5 and pdzk21. A search of GenBank with the sequences ofpdzk5 and pdzk21 revealed that they could be assembled with a partialcDNA sequence, AJ001306, to generate the complete coding sequence ofpdzk5. dbEST sequences from two mouse cDNA clones (GenBank accessionnumbers AA30135 and W50755; IMAGE clone numbers 457904 and 356188)suggested that they might contain the start and stop codons,respectively, of the mouse ortholog of pdzk5. Sequencing of these clonesrevealed that this was indeed the case and confirmed our assignment ofthe translational start and stop codons. The nucleotide sequence forMMSC1 is set forth in SEQ ID NO: 2 with the amino acid sequence of theencoded protein set forth in SEQ ID NO: 3.

[0194]FIG. 1 shows a diagram of MMSC1 indicating the position of the 11PDZ domains and the overlap of the two mouse cDNA clones. FIG. 2 showsan alignment of the first 300 nucleotides of human MMSC1 (H.s._MMSC1)with its translation product (H.s._MMSC1.pep) and the correspondingsequence from the mouse ortholog (M.m._MMSC1; SEQ ID NO: 4), asdetermined from an analysis of the sequence from the above noted clones,with its translation product (M.m._MMSC1.pep; SEQ ID NO: 5). Gaps havebeen introduced into the mouse sequence to optimize the alignment. Thein-frame stop codon at nucleotide 93, shared by the mouse and humansequences, demonstrates that the start codon at nucleotide 115 has beencorrectly identified. The 11 PDZ domains correspond to the amino acidsof MMSC1 as shown in Table 1. TABLE 1 Sequence Correspondence of 11 PDZDomains Domain Number Amino Acid Span 1 133-219 2 247-326 3 364-451 4558-637 5 685-771 6 1067-1158 7 1238-1320 8 1436-1518 9 1532-1613 101675-1760 11 1798-1882

[0195] The nucleotide sequence of MMSC1 was compared with the sequenceof GenBank accession number AJ001306. Other than sequencing errors, thefollowing major differences were noted. First, the AJ001306 is missingat least one exon and possibly more (nucleotide 4492-4575 of SEQ ID NO:2). The absence ofthis exon knocks out PDZ domain number 8. Second, theAJ001306 sequence is either alternatively spliced or unspliced atnucleotide 4770 (relative to SEQ ID NO: 2) which results in a stop codonat nt 4771 (relative to SEQ ID NO: 2). This would knock out PDZ domains9, 10 and 11.

Example 2 MMSC1-Interacting Proteins by Two-Hybrid Analysis

[0196] DNA fragments encoding all or portions of MMSC1 are ligated to atwo-hybrid DNA-binding domain vector such as pGBT.C such that the codingsequence of MMSC1 is in-frame with coding sequence for the Gal4pDNA-binding domain. These DNA fragments may encode specific PDZ domainsof MMSC1 plus the 5 to 10 amino acids N- and C-terminal of each specificPDZ. A plasmid that encodes a DNA-binding domain fusion to a fragment ofMMSC1 PDZ is introduced into the yeast reporter strain (such as J692)along with a library of cDNAs fused to an activation domain.Transformants are spread onto 20-150 mm plates of selective media, suchas yeast minimal media lacking leucine, tryptophan, and histidine, andcontaining 25 mM 3-amino-1,2,4-triazole. After one week incubation at30° C., yeast colonies are assayed for expression of the lacZ reportergene by beta-galactosidase filter assay. Colonies that both grow in theabsence of histidine and are positive for production ofbeta-galactosidase are chosen for further characterization.

[0197] The activation domain plasmid is purified from positive coloniesby the smash-and-grab technique. These plasmids are introduced into E.coli (e.g., DH10B (Gibco BRL)) by electroporation and purified from E.coli by the alkaline lysis method. To test for the specificity of theinteraction, specific activation domain plasmids are cotransformed intostrain J692 with plasmids encoding various DNA-binding domain fusionproteins, including fusions to segments of MMSC1 and human lamin C.Transformants from these experiments are assayed for expression of theHIS3 and lacZ reporter genes. Positives that express reporter genes withMMSC1 constructs and not with lamin C constructs encode bona fideMMSC1-interacting proteins. These proteins are identified andcharacterized by sequence analysis of the insert of the appropriateactivation domain plasmid.

Example 3 Characterization of the Binding Specificity of MMSC1 PDZDomains by Two-Hybrid Analysis

[0198] DNA fragments encoding specific PDZ domains of MMSC1 plus the 5to 10 amino acids N- and C-terminal of each specific PDZ domain aregenerated by PCR amplification. These fragments are ligated to atwo-hybrid DNA-binding domain vector such as pGBT.C such that the codingsequence of MMSC1 is in-frame with coding sequence for the Ga14pDNA-binding domain. An activation domain library is prepared thatencodes an activation domain fused in-frame to random peptide sequencesthat end with a stop codon. An example of this type of library is theClontech random peptide library. A plasmid that encodes a DNA-bindingdomain fusion to a specific MMSC1 PDZ domain is introduced into theyeast reporter strain (such as J692) along with a library of randompeptides fused to an activation domain. Transformants are spread onto20-150 mm plates of selective media, such as yeast minimal media lackingleucine, tryptophan, and histidine, and containing 25 mM3-amino-1,2,4-triazole. After one week incubation at 30° C., yeastcolonies are assayed for expression of the lacZ reporter gene bybeta-galactosidase filter assay. Colonies that both grow in the absenceof histidine and are positive for production of beta-galactosidase arechosen for sequence analysis. The insert of the activation domainconstruct is characterized by sequence analysis. The sequence of thepeptide that binds to the MMSC1 PDZ domain is obtained by conceptualtranslation of the nucleotide sequence. Peptide sequences from multipleisolates are aligned to determine a consensus binding motif. This motifcan be used to identify cellular proteins that bind MMSC1 and to developsmall molecules that inhibit binding to these specific PDZ domains.

Example 4 In vitro Protein-Protein Interaction Assay

[0199] cDNAs encoding each of the MMSC1 PDZ domains (amino acid residuesidentified in Table 1), and any desired control proteins, were generatedby PCR and subcloned as glutathione S-transferase (GST) fusions in pGEXvectors (Pharmacia). After sequencing to confirm expression constructintegrity, the resulting clones were expressed in E. coli and thedesired fusion proteins isolated with glutathione-agarose and recoveredwith glutathione elution. These fusion proteins or control proteins werethen adsorbed to different wells of a 96-well ELISA plate and remainingsites blocked with BSA. Synthetic commercially synthesized peptidesencoding the desired PDZ-binding domain (i.e., the 16 C-terminal aminoacids of MMAC1, or the C-terminal peptide sequences of interactingproteins identified by the approach of Example 2, or the C-terminalpeptide sequences identified by the approach of Example 3), or a controlpeptide, and biotinylated at the amino-terminus, were pre-bound tostreptavidin-alkaline phosphatase in a 4:1 molar ratio. The biotinylatedpeptide streptavidin-alkaline phosphatase complexes were then blockedwith free biotin. These pre-bound peptide streptavidin-alkalinephosphatase complexes were then incubated with the immobilized PDZdomains in wash buffer containing PBS, BSA and triton-X100. Unboundmaterial was removed with repeated washes. Boundpeptide/streptavidin-alkaline phosphatase complex was then quantitatedby a colorimetric phosphatase assay read on a 96-well plate reader.

[0200] The following peptides were used in the initial study:

[0201] SH3 peptide biotin-SGSGILAPPVPPRNTR-COOH (SEQ ID NO: 7)

[0202] AF6 PDZ binding peptide biotin-SGDDGDDPFLQYEFYV-COOH (SEQ ID NO:8)

[0203] MMAC1.388-403 biotin-ENEPFDEDQHTQITKV-COOH (SEQ ID NO: 9).

[0204] The results of the peptide binding ELISA assay is set forth inTable 2. TABLE 2 PDZ Binding Assay MMSc1 PDZ Peptide A405 3 SH3 0.01 AF62.24 MMAC 0.01 5 SH3 0.00 AF6 0.02 MMAC 0.00 6 SH3 0.00 AF6 1.46 MMAC0.23 7 SH3 0.01 AF6 1.05 MMAC 1.25 8 SH3 0.00 AF6 0.29 MMAC 0.30 9 SH30.02 AF6 2.41 MMAC 0.10

[0205] The GST-affinity pull down assay is a complementary in vitromethod for investigating protein-protein interactions. PDZ domain-GSTfusion proteins are incubated with synthetic biotinylated peptides inwash buffer (these peptides were described above). Streptavidin magneticbeads are then added to recover the biotinylated peptide, then unboundmaterials removed by washing. The beads are then incubated with SDS/DTTloading buffer at 100° C. and bound protein detected by SDS/PAGE andcoomasie blue staining.

Example 5 Mutation screening of MMSC1

[0206] Nested PCR amplifications were performed on cDNA from tumor celllines. Total cell line RNAs were reverse transcribed with Superscript II(Life Technologies) and random hexamers. Using the outer primer pairfrom each amplicon (i.e. PDZK5.1A and PDZK5.1P or PDZK5.2A andPDZK5.2P), approximately 10 ng of cDNA from each cell line was amplifiedfor 26 cycles. Products were diluted 60 fold and then reamplified for22-26 cycles using nested M13 tailed primers (i.e. PDZK5.1B and PDZK5.1Qor PDZK5.2B and PDZK5.2Q). Typical primary amplification cyclingconditions were an initial denaturation at 95° for 60s, followed by 26cycles of 96° (12s),58° (15s) and 72° (90s). Typical secondaryamplification cycling conditions were an initial denaturation at 95° for60s, followed by 22-26 cycles of 96° (12s), 58° (15s) and 72° (40s). Theresulting RT-PCR products were sequenced with dye-primer chemistry onABI 377 sequencers. Sequences were examined for the presence of variantsusing the program Sequencher.

[0207] The primers used are set forth in Table 3. The sequence variantsare set forth in Table 4. TABLE 3 Table of Primers Name Primer SequenceSEQ ID NO: PDZK5.1A CAGGTGAGGCAGGGCCGACA 10 PDZK5.1PCTACAGTAGGCAGGGCAACAGG 11 PDZK5.1BGTTTTCCCAGTCACGACGCGGGCTCCCACCTGCTCCTC 12 PDZK5.1QAGGAAACAGCTATGACCATGTGAACACTAACAAACCTTTCC 13 PDZK5.1CGTTTTCCCAGTCACGACGTCAACTCAACCATATACCCTCA 14 PDZK5.1RAGGAAACAGCTATGACCATGGCTGGACATCCTTCACGAAG 15 PDZK5.1DGTTTTCCCAGTCACGACGGCCTTGGATTCAGTGTGGTG 16 PDZK5.1SAGGAAACAGCTATGACCATCCCCAACAAACTGTTTCAGGC 17 PDZK5.1EGTTTTCCCAGTCACGACGCCAGGGAACCAGTCCACACA 18 PDZK5.1TAGGAAACAGCTATGACCATCCTGACTGAATTCCCACAG 19 PDZK5.2ATCCTGGAGGATTAGCAGATCGAG 20 PDZK5.2P GGTAATCCAAAATGCTGAATCCCA 21 PDZK5.2BGTTTTCCCAGTCACGACGAAGATTGGTGGCACAAACGTG 22 PDZK5.2QAGGAAACAGCTATGACCATAGCACTGCCAGGTATTATACTT 23 PDZK5.2CGTTTTCCCAGTCACGACGAGAATTGTTGGCTATGTTGGAAC 24 PDZK5.2RAGGAAACAGCTATGACCATGCTCCAGTTAGAAAGAGAGCTG 25 PDZK5.2DGTTTTCCCAGTCACGACGACATCCTCATCTACTTCTCCA 26 PDZK5.2SAGGAAACAGCTATGACCATAACTCAGCATCATCTGCAATC 27 PDZK5.2EGTTTTCCCAGTCACGACGGGAAAACCTGTTGGGTCCTG 28 PDZK5.2TAGGAAACAGCTATGACCATCGACAGCAAACCAAAGTAAAAGG 29 PDZK5.3AGTGGATTCCTTTGATGGGCACC 30 PDZK5.3P CTTTGAGCCACAACAGGAAGGTC 31 PDZK5.3BGTTTTCCCAGTCACGACGTGAGCTGCTTGAGGTCAATGG 32 PDZK5.3QAGGAAACAGCTATGACCATCTAAAGGGTCCTGGTAATCC 33 PDZK5.3CGTTTTCCCAGTCACGACGCCCCTGAAGTCAAGATTGTTG 34 PDZK5.3RAGGAAACAGCTATGACCATACAACTTTCTTCTTCATTATCTTCC 35 PDZK5.3DGTTTTCCCAGTCACGACGGAAATATTGAAAGCTGTGCC 36 PDZK5.3SAGGAAACAGCTATGACCATGTCAGAAATTCATGCATCTCC 37 PDZK5.3EGTTTTCCCAGTCACGACGAAAGTCTTTCCATTCCCAACAA 38 PDZK5.3TAGGAAACAGCTATGACCATCCATACGGCTGTGCCTCCTG 39 PDZK5.4AGAGTTATATCAAGATCCCTCACCAT 40 PDZK5.4P CAAATATGCTCATGCGTGATCGG 41PDZK5.4B GTTTTCCCAGTCACGACGTTACTTTGGTACACAGTGGTTG 42 PDZK5.4QAGGAAACAGCTATGACCATAAATCTTCTTGCTCCCTCCTT 43 PDZK5.4CGTTTTCCCAGTCACGACGCCCGAATGATGTCCAAGGTCC 44 PDZK5.4RAGGAAACAGCTATGACCATGTCCACCAACAATACTGATCC 45 PDZK5.4DGTTTTCCCAGTCACGACGAGCCACTGGGGTCCACCGAG 46 PDZK5.4SAGGAAACAGCTATGACCATACTCGTGGAGTGGATGACAAAC 47 PDZK5.4EGTTTTCCCAGTCACGACGCAGTTGAGGCCATTAAGAAT 48 PDZK5.4TAGGAAACAGCTATGACCATCAAGTTCAATAATGTGCAGTTCT 49 PDZK5.5ACGCCAATGAAACTTCCTCCTCCT 50 PDZK5.5P TCTCCTGTGAGGCATTTCTCATG 51 PDZK5.5BGTTTTCCCAGTCACGACGCCTTTACCGACCAAAAAATCAGA 52 PDZK5.5QAGGAAACAGCTATGACCATCTGATTGACTGCATCCTCG 53 PDZK5.5CGTTTTCCCAGTCACGACGCATCTGCCATTATTAAGACTGC 54 PDZK5.5RAGGAAACAGCTATGACCATGTGAAGTCTGCATCTGTTGAAT 55 PDZK5.5DGTTTTCCCAGTCACGACGTCCAACAAAAGTCTCCTTCAGT 56 PDZK5.5SAGGAAACAGCTATGACCATAACCTCTAATATCTGGTCACC 57 PDZK5.5EGTTTTCCCAGTCACGACGCTATAGTTATCCATGAAGTCT 58 PDZK5.5TAGGAAACAGCTATGACCATCCGCCTTTCACGATGTCAG 59 PDZK5.6AGAAGGTGCGGCTGGTGGTGTAT 60 PDZK5.6P CTTGCTCTGTCACCCAGGCTG 61 PDZK5.6BGTTTTCCCAGTCACGACGGGCCTGAGCATCGTTGGGAA 62 PDZK5.6QAGGAAACAGCTATGACCATAACCAGGTTTTGCAGGCCAGT 63 PDZK5.6CGTTTTCCCAGTCACGACGTCAGGGTAGTCAGCAGAGTGC 64 PDZK5.6RAGGAAACAGCTATGACCATTACCCACATCCGCGTGAGAC 65

[0208] TABLE 4 +HC,1Ssequence Variants Cell line Type nt variant aachange note MDA-MB-231 breast G1021A gly−>arg Heterozygous variant¹.A172 glioblastoma A1199C glu−>ala Heterozygous variant². A172glioblastoma A1312G ile−>val Heterozygous polymorphism T98G glioblastomaA1312G ile−>val Heterozygous polymorphism T98G glioblastoma A3646Gser−>gly Heterozygous polymorphism NIH OVCAR-3 ovarian A3646G ser−>glyHeterozygous polymorphism MDA-MB-231 breast A3646G ser−>gly Heterozygouspolymorphism NIH OVCAR-3 ovarian A3959G his−>arg Homozygous variant³.U-373MG glioblastoma G4053A none Heterozygous polymorphism U-118MGglioblastoma G4053A none Heterozygous polymorphism T98G glioblastomaG4053A none Heterozygous polymorphism MDA-MB-231 breast G4053A noneHomozygous polymorphism⁴ U-118MG glioblastoma C4192G leu−>valHeterozygous polymorphism T98G glioblastoma C4192G leu−>val Heterozygouspolymorphism NIH OVCAR-3 ovarian C4192G leu−>val Heterozygouspolymorphism HS700T pancreatic C4192G leu−>val Heterozygous polymorphismHS700T pancreatic A4674G none Heterozygous polymorphism splice defect.Both of these possibilities are in accord with the notion that thisamino acid substitution is deleterious.

Example 6 Generation of Polyclonal Antibody Against MMSC1

[0209] Segments of MMSC1 coding sequence are expressed as fusion proteinin E. coli. The overexpressed protein is purified by gel elution andused to immunize rabbits and mice using a procedure similar to the onedescribed by Harlow and Lane, 1988. This procedure has been shown togenerate Abs against various other proteins (for example, see Kraemer etal., 1993).

[0210] Briefly, a stretch of MMSC1 coding sequence is cloned as a fusionprotein in plasmid PET5A (Novagen, Inc., Madison, Wis.). After inductionwith IPTG, the overexpression of a fusion protein with the expectedmolecular weight is verified by SDS/PAGE. Fusion protein is purifiedfrom the gel by electroelution. Identification of the protein as theMMSC1 fusion product is verified by protein sequencing at theN-terminus. Next, the purified protein is used as immunogen in rabbits.Rabbits are immunized with 100 μg of the protein in complete Freund'sadjuvant and boosted twice in 3 week intervals, first with 100 μg ofimmunogen in incomplete Freund's adjuvant followed by 100 μg ofimmunogen in PBS. Antibody containing serum is collected two weeksthereafter. This procedure is repeated to generate antibodies againstthe mutant forms of the MMSC1 gene product. These antibodies, inconjunction with antibodies to wild type MMSC1, are used to detect thepresence and the relative level of the mutant forms in various tissuesand biological fluids.

Example 7 Generation of Polyclonal Antibody Against MMSC1-MMSC1Interacting Protein Complex

[0211] MMSC1 is capable of binding to certain proteins, e.g., MMAC1. Acomplex of the two proteins is prepared, e.g., by mixing purifiedpreparations of each of the two proteins. If desired, the proteincomplex can be stabilized by cross-linking the proteins in the complexby methods known to those of skill in the art. The protein complex isused to immunize rabbits and mice using a procedure similar to the onedescribed by Harlow and Lane, 1988. This procedure has been shown togenerate Abs against various other proteins (for example, see Kraemer etal., 1993).

[0212] Briefly, the purified protein complex is used as immunogen inrabbits. Rabbits are immunized with 100 μg of the protein in completeFreund's adjuvant and boosted twice in 3 week intervals, first with 100μg of immunogen in incomplete Freund's adjuvant followed by 100 μg ofimmunogen in PBS. Antibody containing serum is collected two weeksthereafter.

[0213] This procedure is repeated to generate antibodies against formsof the complex which comprise mutant MMSC1 or mutant MMSC1 interactingprotein (e.g., mutant MMAC1). These antibodies, in conjunction withantibodies to wild type MMSC1 or MMSC1 interacting protein (e.g.,MMAC1), are used to detect the presence and the relative level of themutant forms in various tissues and biological fluids.

Example 8 Generation of Monoclonal Antibodies Specific for MMSC1

[0214] Monoclonal antibodies are generated according to the followingprotocol. Mice are immunized with immunogen comprising intact MMSC1 orMMSC1 peptides (wild type or mutant) conjugated to keyhole limpethemocyanin using glutaraldehyde or EDC as is well known.

[0215] The immunogen is mixed with an adjuvant. Each mouse receives fourinjections of 10 to 100 μg of immunogen and after the fourth injectionblood samples are taken from the mice to determine if the serum containsantibody to the immunogen. Serum titer is determined by ELISA or RIA.Mice with sera indicating the presence of antibody to the immunogen areselected for hybridoma production.

[0216] Spleens are removed from immune mice and a single cell suspensionis prepared (see Harlow and Lane, 1988). Cell fusions are performedessentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3myeloma cells (American Type Culture Collection, Rockville, Md.) arefused with immune spleen cells using polyethylene glycol as described byHarlow and Lane, 1988. Cells are plated at a density of 2×10⁵ cells/wellin 96 well tissue culture plates. Individual wells are examined forgrowth and the supernatants of wells with growth are tested for thepresence of MMSC1 specific antibodies by ELISA or RIA using wild type ormutant MMSC1 target protein. Cells in positive wells are expanded andsubcloned to establish and confirm monoclonality.

[0217] Clones with the desired specificities are expanded and grown asascites in mice or in a hollow fiber system to produce sufficientquantities of antibody for characterization and assay development.

Example 9 Generation of Monoclonal Antibodies Specific for MMSC1-MMSC1Interacting Protein Complex

[0218] Monoclonal antibodies are generated according to the followingprotocol. Mice are immunized with immunogen comprising MMSC1-MMSC1interacting protein complexes (wild type or mutant), such as MMAC1,conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC asis well known. The complexes may be stabilized by cross-linking.

[0219] The immunogen is mixed with an adjuvant. Each mouse receives fourinjections of 10 to 100 μg of immunogen and after the fourth injectionblood samples are taken from the mice to determine if the serum containsantibody to the immunogen. Serum titer is determined by ELISA or RIA.Mice with sera indicating the presence of antibody to the immunogen areselected for hybridoma production.

[0220] Spleens are removed from immune mice and a single cell suspensionis prepared (see Harlow and Lane, 1988). Cell fusions are performedessentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3myeloma cells (American Type Culture Collection, Rockville, Md.) arefused with immune spleen cells using polyethylene glycol as described byHarlow and Lane, 1988. Cells are plated at a density of 2×10⁵ cells/wellin 96 well tissue culture plates. Individual wells are examined forgrowth and the supernatants of wells with growth are tested for thepresence of MMSC1-MMSC1 interacting protein complex specific antibodiesby ELISA or RIA using wild type or mutant MMSC1-MMSC1 interactingprotein complexes as target protein. Cells in positive wells areexpanded and subcloned to establish and confirm monoclonality.

[0221] Clones with the desired specificities are expanded and grown asascites in mice or in a hollow fiber system to produce sufficientquantities of antibody for characterization and assay development.Antibodies are tested for binding to MMSC1 alone or to MMSC1 interactingprotein alone to determine which are specific for the complex as opposedto binding to the individual proteins.

Example 10 Sandwich Assay for MMSC1

[0222] Monoclonal antibody is attached to a solid surface such as aplate, tube, bead or particle. Preferably, the antibody is attached tothe well surface of a 96-well ELISA plate. 100 μL sample (e.g., serum,urine, tissue cytosol) containing the MMSC1 peptide/protein (wild-typeor mutants) is added to the solid phase antibody. The sample isincubated for 2 hrs at room temperature. Next the sample fluid isdecanted, and the solid phase is washed with buffer to remove unboundmaterial. 100 μL of a second monoclonal antibody (to a differentdeterminant on the MMSC1 peptide/protein) is added to the solid phase.This antibody is labeled with a detector molecule (e.g., ¹²⁵I, enzyme,fluorophore, or a chromophore) and the solid phase with the secondantibody is incubated for two hrs at room temperature. The secondantibody is decanted and the solid phase is washed with buffer to removeunbound material.

[0223] The amount of bound label, which is proportional to the amount ofMMSC1 peptide/protein present in the sample, is quantified. Separateassays are performed using monoclonal antibodies which are specific forthe wild-type MMSC1 as well as monoclonal antibodies specific for eachof the mutations identified in MMSC1.

Example 11 Sandwich Assay for MMAC1 Using MMSC1

[0224] MMSC1 or PDZ domain 6 of MMSC1 is attached to a solid surfacesuch as a plate, tube, bead or particle. Preferably, MMSC1 or its PDZdomain is attached to the well surface of a 96-well ELISA plate. 100 μLsample (e.g., serum, urine, tissue cytosol) containing the MMAC1peptide/protein (wild-type or mutants) is added to the solid phaseMMSC1. The sample is incubated for 2 hrs at room temperature. Next thesample fluid is decanted, and the solid phase is washed with buffer toremove unbound material. 100 μL of a monoclonal antibody to MMAC1 isadded to the solid phase. The antibody is labeled with a detectormolecule (e.g., ¹²⁵I, enzyme, fluorophore, or a chromophore) and thesolid phase with the antibody is incubated for two hrs at roomtemperature. The antibody is decanted and the solid phase is washed withbuffer to remove unbound material. The amount of bound label, which isproportional to the amount of wild-type MMAC1 present in the sample, isquantified.

Example 12 Drug Screening

[0225] The invention is useful in screening for drugs which can overcomemutations in MMSC1 and also mutations in MMAC1. The knowledge that MMSC1and MMAC1 form a complex is useful in designing such assays. If amutation is present in either MMSC1 or in MMAC1 which prevents theMMSC1-MMAC1 complex from forming, drugs may be screened which willovercome the mutation and allow the protein complex to form and to beactive. Such screening assays can be, e.g., a yeast two hybrid assaywhich is dependent upon two proteins interacting. In such an assay, thepresence of a mutant protein may show no activity or low activity insuch an assay, while the presence of a useful drug will result information of a proper complex which results in activity in the assay.

[0226] A simple binding assay which shows the binding, i.e., formationof a complex, can similarly be used as outlined above. Useful drugs willincrease the formation of MMSC1-MMAC1 complexes. Antibodies may also beused to monitor the amount of complex present. Antibodies specific forthe complex are especially useful. If the presence of a drug increasesthe amount of complex present then the drug is a good candidate fortreating the cancer which is a result of the mutation in either theMMSC1 or the MMAC1.

[0227] While the invention has been disclosed in this patent applicationby reference to the details of preferred embodiments of the invention,it is to be understood that the disclosure is intended in anillustrative rather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

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[0430] U.S. Pat. No. 5,455,166.

[0431] U.S. Pat. No. 5,550,050

1 65 1 4 PRT Artificial Sequence Description of Artificial SequencePDZConsensus Domain 1 Glu Xaa Xaa Xaa 1 2 5836 DNA Homo sapiens CDS(115)..(5757) 2 ctcacttccg cccaggtgag gcagggccga caccgagccc gcccgacccgggctcccacc 60 tgctcctcca gcgcaccagg tgtctttaag agtgattgaa gagaataattcaaa atg 117 Met 1 cct gaa aat cct gct aca gat aaa ctg cag gtg ctg caggta ctt gat 165 Pro Glu Asn Pro Ala Thr Asp Lys Leu Gln Val Leu Gln ValLeu Asp 5 10 15 cgc ctg aaa atg aaa ttg cag gag aag ggt gac acg tcg cagaat gag 213 Arg Leu Lys Met Lys Leu Gln Glu Lys Gly Asp Thr Ser Gln AsnGlu 20 25 30 aag tta tct atg ttt tat gag aca cta aag agt cct ctc ttc aaccag 261 Lys Leu Ser Met Phe Tyr Glu Thr Leu Lys Ser Pro Leu Phe Asn Gln35 40 45 ata ctc aca ctt cag cag tcc atc aag caa ctg aag ggt caa ctc aac309 Ile Leu Thr Leu Gln Gln Ser Ile Lys Gln Leu Lys Gly Gln Leu Asn 5055 60 65 cat ata ccc tca gat tgt tca gcc aac ttt gat ttt tct agg aaa ggt357 His Ile Pro Ser Asp Cys Ser Ala Asn Phe Asp Phe Ser Arg Lys Gly 7075 80 ttg tta gtg ttc aca gat ggt tcc att act aat gga aat gtc cac agg405 Leu Leu Val Phe Thr Asp Gly Ser Ile Thr Asn Gly Asn Val His Arg 8590 95 ccc tct aat aac tcg act gta tct ggg tta ttt ccg tgg acc ccg aag453 Pro Ser Asn Asn Ser Thr Val Ser Gly Leu Phe Pro Trp Thr Pro Lys 100105 110 ttg gga aat gaa gac ttt aac tca gtc att caa cag atg gct cag ggc501 Leu Gly Asn Glu Asp Phe Asn Ser Val Ile Gln Gln Met Ala Gln Gly 115120 125 cgg caa att gaa tat ata gat ata gaa cgg cct tca act gga ggc ctt549 Arg Gln Ile Glu Tyr Ile Asp Ile Glu Arg Pro Ser Thr Gly Gly Leu 130135 140 145 gga ttc agt gtg gtg gcc ctc aga agt caa aat ctc gga aaa gttgat 597 Gly Phe Ser Val Val Ala Leu Arg Ser Gln Asn Leu Gly Lys Val Asp150 155 160 atc ttc gtg aag gat gtc cag cca ggg agt gta gca gac agg gatcaa 645 Ile Phe Val Lys Asp Val Gln Pro Gly Ser Val Ala Asp Arg Asp Gln165 170 175 aga tta aag gaa aat gat caa ata ttg gcc att aat cac acg ccattg 693 Arg Leu Lys Glu Asn Asp Gln Ile Leu Ala Ile Asn His Thr Pro Leu180 185 190 gat cag aac att tcc cat cag caa gca att gca tta tta caa caaacc 741 Asp Gln Asn Ile Ser His Gln Gln Ala Ile Ala Leu Leu Gln Gln Thr195 200 205 act gga tct ttg aga ctg att gtg gcc agg gaa cca gtc cac acaaaa 789 Thr Gly Ser Leu Arg Leu Ile Val Ala Arg Glu Pro Val His Thr Lys210 215 220 225 agc agt act tct agc agc cta aat gat aca act ctg cct gaaaca gtt 837 Ser Ser Thr Ser Ser Ser Leu Asn Asp Thr Thr Leu Pro Glu ThrVal 230 235 240 tgt tgg ggc cat gtt gaa gag gtt gag ctc att aat gat ggctct gga 885 Cys Trp Gly His Val Glu Glu Val Glu Leu Ile Asn Asp Gly SerGly 245 250 255 cta ggt ttt gga ata gtt gga gga aaa aca agt ggc gtg gttgtg agg 933 Leu Gly Phe Gly Ile Val Gly Gly Lys Thr Ser Gly Val Val ValArg 260 265 270 act ata gtt cct gga gga tta gca gat cga gat gga aga ctccag aca 981 Thr Ile Val Pro Gly Gly Leu Ala Asp Arg Asp Gly Arg Leu GlnThr 275 280 285 ggg gac cac atc ttg aag att ggt ggc aca aac gtg cag ggaatg acc 1029 Gly Asp His Ile Leu Lys Ile Gly Gly Thr Asn Val Gln Gly MetThr 290 295 300 305 agt gag caa gtt gca caa gtt cta agg aac tgt ggg aattca gtc agg 1077 Ser Glu Gln Val Ala Gln Val Leu Arg Asn Cys Gly Asn SerVal Arg 310 315 320 atg ctc gtt gct aga gat cca gct ggt gac att tca gtcacc ccc cct 1125 Met Leu Val Ala Arg Asp Pro Ala Gly Asp Ile Ser Val ThrPro Pro 325 330 335 gcc cct gca gcc tta cct gtt gcc ctg cct act gta gccagc aag ggc 1173 Ala Pro Ala Ala Leu Pro Val Ala Leu Pro Thr Val Ala SerLys Gly 340 345 350 cct ggt tct gac agt tct ctt ttt gaa act tat aat gttgag ctt gtg 1221 Pro Gly Ser Asp Ser Ser Leu Phe Glu Thr Tyr Asn Val GluLeu Val 355 360 365 aga aaa gat ggg cag agt ctt gga att aga att gtt ggctat gtt gga 1269 Arg Lys Asp Gly Gln Ser Leu Gly Ile Arg Ile Val Gly TyrVal Gly 370 375 380 385 aca tct cat aca ggg gaa gct tca ggg att tat gtgaaa agt gta ata 1317 Thr Ser His Thr Gly Glu Ala Ser Gly Ile Tyr Val LysSer Val Ile 390 395 400 cct ggc agt gct gcg tac cac aat ggc cac att caagtg aat gac aaa 1365 Pro Gly Ser Ala Ala Tyr His Asn Gly His Ile Gln ValAsn Asp Lys 405 410 415 ata gtt gct gtc gat ggc gtg aac att cag ggt tttgcc aac cat gat 1413 Ile Val Ala Val Asp Gly Val Asn Ile Gln Gly Phe AlaAsn His Asp 420 425 430 gtt gtt gaa gta tta cga aat gca ggg cag gtg gtacac cta acc cta 1461 Val Val Glu Val Leu Arg Asn Ala Gly Gln Val Val HisLeu Thr Leu 435 440 445 gtt cga agg aag aca tcc tca tct act tct cca cttgaa cca cct tca 1509 Val Arg Arg Lys Thr Ser Ser Ser Thr Ser Pro Leu GluPro Pro Ser 450 455 460 465 gac aga gga act gtt gta gaa cca ctg aaa ccacca gct ctc ttt cta 1557 Asp Arg Gly Thr Val Val Glu Pro Leu Lys Pro ProAla Leu Phe Leu 470 475 480 act gga gca gtg gaa act gaa act aat gtg gatggt gaa gat gag gaa 1605 Thr Gly Ala Val Glu Thr Glu Thr Asn Val Asp GlyGlu Asp Glu Glu 485 490 495 att aaa gaa aga att gat act tta aaa aat gacaac ata caa gcc tta 1653 Ile Lys Glu Arg Ile Asp Thr Leu Lys Asn Asp AsnIle Gln Ala Leu 500 505 510 gaa aaa ttg gaa aaa gtc cca gac tct cca gaaaat gag ctg aaa tcc 1701 Glu Lys Leu Glu Lys Val Pro Asp Ser Pro Glu AsnGlu Leu Lys Ser 515 520 525 aga tgg gaa aac ctg ttg ggt cct gat tat gaagta atg gtt gct act 1749 Arg Trp Glu Asn Leu Leu Gly Pro Asp Tyr Glu ValMet Val Ala Thr 530 535 540 545 ttg gac aca cag att gca gat gat gct gagtta cag aaa tat tca aag 1797 Leu Asp Thr Gln Ile Ala Asp Asp Ala Glu LeuGln Lys Tyr Ser Lys 550 555 560 ctg ctg cct att cac act ctg agg ctt ggtgtg gaa gtg gat tcc ttt 1845 Leu Leu Pro Ile His Thr Leu Arg Leu Gly ValGlu Val Asp Ser Phe 565 570 575 gat ggg cac cat tat att tct tca att gtttct ggt ggt cct gtt gat 1893 Asp Gly His His Tyr Ile Ser Ser Ile Val SerGly Gly Pro Val Asp 580 585 590 aca ttg ggt ctc cta cag cca gaa gat gagctg ctt gag gtc aat ggc 1941 Thr Leu Gly Leu Leu Gln Pro Glu Asp Glu LeuLeu Glu Val Asn Gly 595 600 605 atg cag ctt tat gga aaa tct cgc cga gaagca gtc tcc ttt ctt aaa 1989 Met Gln Leu Tyr Gly Lys Ser Arg Arg Glu AlaVal Ser Phe Leu Lys 610 615 620 625 gaa gtg cca ccc cct ttt act ttg gtttgc tgt cgg agg ttg ttt gat 2037 Glu Val Pro Pro Pro Phe Thr Leu Val CysCys Arg Arg Leu Phe Asp 630 635 640 gat gaa gct tct gta gat gaa cca aggcgc act gaa acc tct ctt cct 2085 Asp Glu Ala Ser Val Asp Glu Pro Arg ArgThr Glu Thr Ser Leu Pro 645 650 655 gag aca gag gtt gac cac aat atg gatgtc aat act gaa gaa gat gat 2133 Glu Thr Glu Val Asp His Asn Met Asp ValAsn Thr Glu Glu Asp Asp 660 665 670 gat ggg gaa tta gca ctg tgg tcc cctgaa gtc aag att gtt gaa cta 2181 Asp Gly Glu Leu Ala Leu Trp Ser Pro GluVal Lys Ile Val Glu Leu 675 680 685 gta aaa gat tgt aaa ggt ttg gga ttcagc att ttg gat tac cag gac 2229 Val Lys Asp Cys Lys Gly Leu Gly Phe SerIle Leu Asp Tyr Gln Asp 690 695 700 705 cct tta gat cct aca aga tca gtgatt gtg atc cgc tcc ctg gta gca 2277 Pro Leu Asp Pro Thr Arg Ser Val IleVal Ile Arg Ser Leu Val Ala 710 715 720 gat ggt gta gca gaa aga agt ggggga cta tta cct gga gac cgc ctg 2325 Asp Gly Val Ala Glu Arg Ser Gly GlyLeu Leu Pro Gly Asp Arg Leu 725 730 735 gtc tca gtc aat gaa tac tgt ttggac aac acc tca ctt gct gaa gct 2373 Val Ser Val Asn Glu Tyr Cys Leu AspAsn Thr Ser Leu Ala Glu Ala 740 745 750 gtg gaa ata ttg aaa gct gtg ccacca ggc cta gta cac ctt ggc atc 2421 Val Glu Ile Leu Lys Ala Val Pro ProGly Leu Val His Leu Gly Ile 755 760 765 tgt aag cct ttg gtg gaa gat aatgaa gaa gaa agt tgt tat att tta 2469 Cys Lys Pro Leu Val Glu Asp Asn GluGlu Glu Ser Cys Tyr Ile Leu 770 775 780 785 cat tca agc agt aat gaa gacaag act gaa ttt tca gga aca att cat 2517 His Ser Ser Ser Asn Glu Asp LysThr Glu Phe Ser Gly Thr Ile His 790 795 800 gat ata aat tca tct tta atactc gaa gca ccc aag gga ttt aga gat 2565 Asp Ile Asn Ser Ser Leu Ile LeuGlu Ala Pro Lys Gly Phe Arg Asp 805 810 815 gaa cca tat ttt aaa gaa gaactt gtg gat gaa cca ttt cta gat ctg 2613 Glu Pro Tyr Phe Lys Glu Glu LeuVal Asp Glu Pro Phe Leu Asp Leu 820 825 830 gga aag tct ttc cat tcc caacaa aaa gag ata gag caa agc aag gag 2661 Gly Lys Ser Phe His Ser Gln GlnLys Glu Ile Glu Gln Ser Lys Glu 835 840 845 gcc tgg gag atg cat gaa tttctg act cct aga ttg cag gaa atg gat 2709 Ala Trp Glu Met His Glu Phe LeuThr Pro Arg Leu Gln Glu Met Asp 850 855 860 865 gaa gaa aga gaa atg cttgtt gat gaa gaa tat gag tta tat caa gat 2757 Glu Glu Arg Glu Met Leu ValAsp Glu Glu Tyr Glu Leu Tyr Gln Asp 870 875 880 ccc tca cca tcc atg gagttg tat ccc ttg tcg cac att caa gag gcc 2805 Pro Ser Pro Ser Met Glu LeuTyr Pro Leu Ser His Ile Gln Glu Ala 885 890 895 act cct gtg ccc tct gtgaat gaa ctt cac ttt ggt aca cag tgg ttg 2853 Thr Pro Val Pro Ser Val AsnGlu Leu His Phe Gly Thr Gln Trp Leu 900 905 910 cat gat aat gaa cca tccgag tct caa gag gca aga acc ggg agg act 2901 His Asp Asn Glu Pro Ser GluSer Gln Glu Ala Arg Thr Gly Arg Thr 915 920 925 gtc tat tcc cag gag gcacag ccg tat ggc tat tgc cct gaa aat gtg 2949 Val Tyr Ser Gln Glu Ala GlnPro Tyr Gly Tyr Cys Pro Glu Asn Val 930 935 940 945 atg aaa gaa aat tttgtc atg gag tcc cta cca tct gta cca tca act 2997 Met Lys Glu Asn Phe ValMet Glu Ser Leu Pro Ser Val Pro Ser Thr 950 955 960 gaa gga aac agt caacaa ggc aga ttt gac gac ctg gaa aat ctt aat 3045 Glu Gly Asn Ser Gln GlnGly Arg Phe Asp Asp Leu Glu Asn Leu Asn 965 970 975 tca tta gca aaa actagt ctg gat tta ggc atg atc ccg aat gat gtc 3093 Ser Leu Ala Lys Thr SerLeu Asp Leu Gly Met Ile Pro Asn Asp Val 980 985 990 caa ggt cct agc ttgctc att gac ctt cct gtt gtg gct caa agg agg 3141 Gln Gly Pro Ser Leu LeuIle Asp Leu Pro Val Val Ala Gln Arg Arg 995 1000 1005 gag caa gaa gatttg cct tta tat caa cac caa gcg aca cga gtt att 3189 Glu Gln Glu Asp LeuPro Leu Tyr Gln His Gln Ala Thr Arg Val Ile 1010 1015 1020 1025 tcc aaggcc tca gca tac aca gga atg ttg tct tct aga tat gcc act 3237 Ser Lys AlaSer Ala Tyr Thr Gly Met Leu Ser Ser Arg Tyr Ala Thr 1030 1035 1040 gataca tgt gag tta cct gag aga gaa gaa ggc gaa gga gaa gaa act 3285 Asp ThrCys Glu Leu Pro Glu Arg Glu Glu Gly Glu Gly Glu Glu Thr 1045 1050 1055cca aat ttt agc cac tgg ggt cca ccg aga att gtt gag att ttt aga 3333 ProAsn Phe Ser His Trp Gly Pro Pro Arg Ile Val Glu Ile Phe Arg 1060 10651070 gaa ccc aat gtg tct ctt ggg atc agt att gtt ggt gga caa act gtt3381 Glu Pro Asn Val Ser Leu Gly Ile Ser Ile Val Gly Gly Gln Thr Val1075 1080 1085 ata aaa cgt cta aag aat gga gag gag ctt aaa ggt ata ttcatc aaa 3429 Ile Lys Arg Leu Lys Asn Gly Glu Glu Leu Lys Gly Ile Phe IleLys 1090 1095 1100 1105 caa gtt tta gaa gac agt cca gca ggg aag acg aacgca ctt aaa act 3477 Gln Val Leu Glu Asp Ser Pro Ala Gly Lys Thr Asn AlaLeu Lys Thr 1110 1115 1120 gga gat aaa ata ctt gag gtg tct gga gta gatttg cag aat gcc tca 3525 Gly Asp Lys Ile Leu Glu Val Ser Gly Val Asp LeuGln Asn Ala Ser 1125 1130 1135 cac agc gaa gca gtt gag gcc att aag aatgca gga aac cct gtg gtg 3573 His Ser Glu Ala Val Glu Ala Ile Lys Asn AlaGly Asn Pro Val Val 1140 1145 1150 ttc att gtt cag agt ttg tca tcc actcca cga gtc att cct aat gta 3621 Phe Ile Val Gln Ser Leu Ser Ser Thr ProArg Val Ile Pro Asn Val 1155 1160 1165 cat aac aag gcc aac aaa atc accagt aac cag aac cag gac acc caa 3669 His Asn Lys Ala Asn Lys Ile Thr SerAsn Gln Asn Gln Asp Thr Gln 1170 1175 1180 1185 gaa aag aaa gaa aag aggcaa gga act gct cca ccg cca atg aaa ctt 3717 Glu Lys Lys Glu Lys Arg GlnGly Thr Ala Pro Pro Pro Met Lys Leu 1190 1195 1200 cct cct cct tat aaagct ctg act gat gac agt gat gaa aat gaa gaa 3765 Pro Pro Pro Tyr Lys AlaLeu Thr Asp Asp Ser Asp Glu Asn Glu Glu 1205 1210 1215 gaa gat gcc tttacc gac caa aaa atc aga caa aga tat gca gat ctg 3813 Glu Asp Ala Phe ThrAsp Gln Lys Ile Arg Gln Arg Tyr Ala Asp Leu 1220 1225 1230 cct gga gaactg cac att att gaa ctt gaa aaa gat aag aat gga ctt 3861 Pro Gly Glu LeuHis Ile Ile Glu Leu Glu Lys Asp Lys Asn Gly Leu 1235 1240 1245 gga ctcagc ctt gct ggt aat aaa gac cga tca cgc atg agc ata ttt 3909 Gly Leu SerLeu Ala Gly Asn Lys Asp Arg Ser Arg Met Ser Ile Phe 1250 1255 1260 1265gtg gtg gga att aac ccg gaa gga cct gct gcc gca gat gga cga atg 3957 ValVal Gly Ile Asn Pro Glu Gly Pro Ala Ala Ala Asp Gly Arg Met 1270 12751280 cat att gga gat gaa ctc tta gag ata aac aat cag att ctg tat gga4005 His Ile Gly Asp Glu Leu Leu Glu Ile Asn Asn Gln Ile Leu Tyr Gly1285 1290 1295 aga agt cac caa aat gca tct gcc att att aag act gcc ccatca aag 4053 Arg Ser His Gln Asn Ala Ser Ala Ile Ile Lys Thr Ala Pro SerLys 1300 1305 1310 gtc aag ctg gtt ttc atc aga aac gag gat gca gtc aatcag atg gcc 4101 Val Lys Leu Val Phe Ile Arg Asn Glu Asp Ala Val Asn GlnMet Ala 1315 1320 1325 gtt act ccc ttt cca gtg cca tca agt tct cca tcttct att gag gat 4149 Val Thr Pro Phe Pro Val Pro Ser Ser Ser Pro Ser SerIle Glu Asp 1330 1335 1340 1345 cag agc ggc acc gaa cct att agt agt gaggaa gat ggc agc ctc gaa 4197 Gln Ser Gly Thr Glu Pro Ile Ser Ser Glu GluAsp Gly Ser Leu Glu 1350 1355 1360 gtt ggt att aaa caa ttg cct gaa agtgaa agc ttc aaa ctg gct gtc 4245 Val Gly Ile Lys Gln Leu Pro Glu Ser GluSer Phe Lys Leu Ala Val 1365 1370 1375 agc cag atg aaa cag caa aaa tatcca aca aaa gtc tcc ttc agt tca 4293 Ser Gln Met Lys Gln Gln Lys Tyr ProThr Lys Val Ser Phe Ser Ser 1380 1385 1390 caa gag ata cca tta gca ccagct tca tca tac cat tca aca gat gca 4341 Gln Glu Ile Pro Leu Ala Pro AlaSer Ser Tyr His Ser Thr Asp Ala 1395 1400 1405 gac ttc aca ggc tat ggtggt ttc cag gct cct ctg tca gtg gac ccc 4389 Asp Phe Thr Gly Tyr Gly GlyPhe Gln Ala Pro Leu Ser Val Asp Pro 1410 1415 1420 1425 gca acg tgt cccatt gtc cct gga cag gaa atg att ata gaa ata tcc 4437 Ala Thr Cys Pro IleVal Pro Gly Gln Glu Met Ile Ile Glu Ile Ser 1430 1435 1440 aag gga cgttca ggg ctt ggt ctc agc att gtg gga gga aaa gac aca 4485 Lys Gly Arg SerGly Leu Gly Leu Ser Ile Val Gly Gly Lys Asp Thr 1445 1450 1455 ccc ttgaat gct ata gtt atc cat gaa gtc tat gaa gaa ggg gca gca 4533 Pro Leu AsnAla Ile Val Ile His Glu Val Tyr Glu Glu Gly Ala Ala 1460 1465 1470 gccaga gat gga aga ctt tgg gct ggt gac cag ata tta gag gtt aat 4581 Ala ArgAsp Gly Arg Leu Trp Ala Gly Asp Gln Ile Leu Glu Val Asn 1475 1480 1485ggg gtt gac ctg agg aac tcc agc cac gaa gaa gcc atc aca gcc ctg 4629 GlyVal Asp Leu Arg Asn Ser Ser His Glu Glu Ala Ile Thr Ala Leu 1490 14951500 1505 agg cag acc ccc cag aag gtg cgg ctg gtg gtg tat aga gat gaagca 4677 Arg Gln Thr Pro Gln Lys Val Arg Leu Val Val Tyr Arg Asp Glu Ala1510 1515 1520 cac tac cgg gat gag gag aac ttg gag att ttc cct gtg gatctg cag 4725 His Tyr Arg Asp Glu Glu Asn Leu Glu Ile Phe Pro Val Asp LeuGln 1525 1530 1535 aag aaa gct ggc cgg ggc ctg ggc ctg agc atc gtt gggaaa cga aat 4773 Lys Lys Ala Gly Arg Gly Leu Gly Leu Ser Ile Val Gly LysArg Asn 1540 1545 1550 gga agc gga gtg ttt att tct gac atc gtg aaa ggcgga gcc gca gac 4821 Gly Ser Gly Val Phe Ile Ser Asp Ile Val Lys Gly GlyAla Ala Asp 1555 1560 1565 ctg gat ggg aga ttg att cag gga gat cag atctta tct gtg aat ggg 4869 Leu Asp Gly Arg Leu Ile Gln Gly Asp Gln Ile LeuSer Val Asn Gly 1570 1575 1580 1585 gag gac atg aga aat gcc tca cag gagaca gtg gcc acc atc ctc aag 4917 Glu Asp Met Arg Asn Ala Ser Gln Glu ThrVal Ala Thr Ile Leu Lys 1590 1595 1600 tgt gca cag gga ctt gtg cag ctagag att gga aga ctc cga gct ggt 4965 Cys Ala Gln Gly Leu Val Gln Leu GluIle Gly Arg Leu Arg Ala Gly 1605 1610 1615 tcc tgg acc tcc gca agg acgaca tca cag aac agt cag ggt agt cag 5013 Ser Trp Thr Ser Ala Arg Thr ThrSer Gln Asn Ser Gln Gly Ser Gln 1620 1625 1630 cag agt gca cac agc agctgt cat ccc tcc ttc gct cct gtc atc act 5061 Gln Ser Ala His Ser Ser CysHis Pro Ser Phe Ala Pro Val Ile Thr 1635 1640 1645 ggc ctg caa aac ctggtt ggc aca aaa aga gtt tca gat cct tcc cag 5109 Gly Leu Gln Asn Leu ValGly Thr Lys Arg Val Ser Asp Pro Ser Gln 1650 1655 1660 1665 aaa aat tcaggc aca gat atg gaa cca agg act gtt gag ata aac agg 5157 Lys Asn Ser GlyThr Asp Met Glu Pro Arg Thr Val Glu Ile Asn Arg 1670 1675 1680 gag ctcagt gat gcc ctt gga atc agt att gct gga gga aga gga agt 5205 Glu Leu SerAsp Ala Leu Gly Ile Ser Ile Ala Gly Gly Arg Gly Ser 1685 1690 1695 ccctta gga gat atc ccc gta ttt att gcc atg att cag gct agc gga 5253 Pro LeuGly Asp Ile Pro Val Phe Ile Ala Met Ile Gln Ala Ser Gly 1700 1705 1710gtg gcc gca cgg aca cag aag ctt aaa gtt gga gat cgg att gtc agc 5301 ValAla Ala Arg Thr Gln Lys Leu Lys Val Gly Asp Arg Ile Val Ser 1715 17201725 att aac ggg caa cct ttg gat ggg ctg tct cac gcg gat gtg gtt aat5349 Ile Asn Gly Gln Pro Leu Asp Gly Leu Ser His Ala Asp Val Val Asn1730 1735 1740 1745 ctg ctg aag aat gcc tac ggg cgc att atc ctg cag gttgta gca gat 5397 Leu Leu Lys Asn Ala Tyr Gly Arg Ile Ile Leu Gln Val ValAla Asp 1750 1755 1760 acc aat ata agc gcc ata gca gct cag ctt gaa aacatg tct aca ggc 5445 Thr Asn Ile Ser Ala Ile Ala Ala Gln Leu Glu Asn MetSer Thr Gly 1765 1770 1775 tac cac ctt ggt tcg ccc act gct gaa cac catcca gaa gac aca gaa 5493 Tyr His Leu Gly Ser Pro Thr Ala Glu His His ProGlu Asp Thr Glu 1780 1785 1790 aca cct cca cct aag att att act ttg gagaaa ggc tct gaa ggc ttg 5541 Thr Pro Pro Pro Lys Ile Ile Thr Leu Glu LysGly Ser Glu Gly Leu 1795 1800 1805 ggg ttt agt att gta ggg ggt tat ggaagt ccc cat gga gac ctg cca 5589 Gly Phe Ser Ile Val Gly Gly Tyr Gly SerPro His Gly Asp Leu Pro 1810 1815 1820 1825 att tat gtc aag act gta tttgca aag gga gca gct gca gat gac ggc 5637 Ile Tyr Val Lys Thr Val Phe AlaLys Gly Ala Ala Ala Asp Asp Gly 1830 1835 1840 cga tta aaa cga ggg gatcag att tta gct gtt aat ggc gag acc ctg 5685 Arg Leu Lys Arg Gly Asp GlnIle Leu Ala Val Asn Gly Glu Thr Leu 1845 1850 1855 gaa ggt gtt act catgag caa gca gtc gcc att cta aaa cac cag aga 5733 Glu Gly Val Thr His GluGln Ala Val Ala Ile Leu Lys His Gln Arg 1860 1865 1870 ggg act gta acctta act gtg ctg tcatgagcct cgggcctgat cacaagatag 5787 Gly Thr Val ThrLeu Thr Val Leu 1875 1880 atgttgttgt ttagaatatc cacaggcaga tgaagttctgagtgggtat 5836 3 1881 PRT Homo sapiens 3 Met Pro Glu Asn Pro Ala Thr AspLys Leu Gln Val Leu Gln Val Leu 1 5 10 15 Asp Arg Leu Lys Met Lys LeuGln Glu Lys Gly Asp Thr Ser Gln Asn 20 25 30 Glu Lys Leu Ser Met Phe TyrGlu Thr Leu Lys Ser Pro Leu Phe Asn 35 40 45 Gln Ile Leu Thr Leu Gln GlnSer Ile Lys Gln Leu Lys Gly Gln Leu 50 55 60 Asn His Ile Pro Ser Asp CysSer Ala Asn Phe Asp Phe Ser Arg Lys 65 70 75 80 Gly Leu Leu Val Phe ThrAsp Gly Ser Ile Thr Asn Gly Asn Val His 85 90 95 Arg Pro Ser Asn Asn SerThr Val Ser Gly Leu Phe Pro Trp Thr Pro 100 105 110 Lys Leu Gly Asn GluAsp Phe Asn Ser Val Ile Gln Gln Met Ala Gln 115 120 125 Gly Arg Gln IleGlu Tyr Ile Asp Ile Glu Arg Pro Ser Thr Gly Gly 130 135 140 Leu Gly PheSer Val Val Ala Leu Arg Ser Gln Asn Leu Gly Lys Val 145 150 155 160 AspIle Phe Val Lys Asp Val Gln Pro Gly Ser Val Ala Asp Arg Asp 165 170 175Gln Arg Leu Lys Glu Asn Asp Gln Ile Leu Ala Ile Asn His Thr Pro 180 185190 Leu Asp Gln Asn Ile Ser His Gln Gln Ala Ile Ala Leu Leu Gln Gln 195200 205 Thr Thr Gly Ser Leu Arg Leu Ile Val Ala Arg Glu Pro Val His Thr210 215 220 Lys Ser Ser Thr Ser Ser Ser Leu Asn Asp Thr Thr Leu Pro GluThr 225 230 235 240 Val Cys Trp Gly His Val Glu Glu Val Glu Leu Ile AsnAsp Gly Ser 245 250 255 Gly Leu Gly Phe Gly Ile Val Gly Gly Lys Thr SerGly Val Val Val 260 265 270 Arg Thr Ile Val Pro Gly Gly Leu Ala Asp ArgAsp Gly Arg Leu Gln 275 280 285 Thr Gly Asp His Ile Leu Lys Ile Gly GlyThr Asn Val Gln Gly Met 290 295 300 Thr Ser Glu Gln Val Ala Gln Val LeuArg Asn Cys Gly Asn Ser Val 305 310 315 320 Arg Met Leu Val Ala Arg AspPro Ala Gly Asp Ile Ser Val Thr Pro 325 330 335 Pro Ala Pro Ala Ala LeuPro Val Ala Leu Pro Thr Val Ala Ser Lys 340 345 350 Gly Pro Gly Ser AspSer Ser Leu Phe Glu Thr Tyr Asn Val Glu Leu 355 360 365 Val Arg Lys AspGly Gln Ser Leu Gly Ile Arg Ile Val Gly Tyr Val 370 375 380 Gly Thr SerHis Thr Gly Glu Ala Ser Gly Ile Tyr Val Lys Ser Val 385 390 395 400 IlePro Gly Ser Ala Ala Tyr His Asn Gly His Ile Gln Val Asn Asp 405 410 415Lys Ile Val Ala Val Asp Gly Val Asn Ile Gln Gly Phe Ala Asn His 420 425430 Asp Val Val Glu Val Leu Arg Asn Ala Gly Gln Val Val His Leu Thr 435440 445 Leu Val Arg Arg Lys Thr Ser Ser Ser Thr Ser Pro Leu Glu Pro Pro450 455 460 Ser Asp Arg Gly Thr Val Val Glu Pro Leu Lys Pro Pro Ala LeuPhe 465 470 475 480 Leu Thr Gly Ala Val Glu Thr Glu Thr Asn Val Asp GlyGlu Asp Glu 485 490 495 Glu Ile Lys Glu Arg Ile Asp Thr Leu Lys Asn AspAsn Ile Gln Ala 500 505 510 Leu Glu Lys Leu Glu Lys Val Pro Asp Ser ProGlu Asn Glu Leu Lys 515 520 525 Ser Arg Trp Glu Asn Leu Leu Gly Pro AspTyr Glu Val Met Val Ala 530 535 540 Thr Leu Asp Thr Gln Ile Ala Asp AspAla Glu Leu Gln Lys Tyr Ser 545 550 555 560 Lys Leu Leu Pro Ile His ThrLeu Arg Leu Gly Val Glu Val Asp Ser 565 570 575 Phe Asp Gly His His TyrIle Ser Ser Ile Val Ser Gly Gly Pro Val 580 585 590 Asp Thr Leu Gly LeuLeu Gln Pro Glu Asp Glu Leu Leu Glu Val Asn 595 600 605 Gly Met Gln LeuTyr Gly Lys Ser Arg Arg Glu Ala Val Ser Phe Leu 610 615 620 Lys Glu ValPro Pro Pro Phe Thr Leu Val Cys Cys Arg Arg Leu Phe 625 630 635 640 AspAsp Glu Ala Ser Val Asp Glu Pro Arg Arg Thr Glu Thr Ser Leu 645 650 655Pro Glu Thr Glu Val Asp His Asn Met Asp Val Asn Thr Glu Glu Asp 660 665670 Asp Asp Gly Glu Leu Ala Leu Trp Ser Pro Glu Val Lys Ile Val Glu 675680 685 Leu Val Lys Asp Cys Lys Gly Leu Gly Phe Ser Ile Leu Asp Tyr Gln690 695 700 Asp Pro Leu Asp Pro Thr Arg Ser Val Ile Val Ile Arg Ser LeuVal 705 710 715 720 Ala Asp Gly Val Ala Glu Arg Ser Gly Gly Leu Leu ProGly Asp Arg 725 730 735 Leu Val Ser Val Asn Glu Tyr Cys Leu Asp Asn ThrSer Leu Ala Glu 740 745 750 Ala Val Glu Ile Leu Lys Ala Val Pro Pro GlyLeu Val His Leu Gly 755 760 765 Ile Cys Lys Pro Leu Val Glu Asp Asn GluGlu Glu Ser Cys Tyr Ile 770 775 780 Leu His Ser Ser Ser Asn Glu Asp LysThr Glu Phe Ser Gly Thr Ile 785 790 795 800 His Asp Ile Asn Ser Ser LeuIle Leu Glu Ala Pro Lys Gly Phe Arg 805 810 815 Asp Glu Pro Tyr Phe LysGlu Glu Leu Val Asp Glu Pro Phe Leu Asp 820 825 830 Leu Gly Lys Ser PheHis Ser Gln Gln Lys Glu Ile Glu Gln Ser Lys 835 840 845 Glu Ala Trp GluMet His Glu Phe Leu Thr Pro Arg Leu Gln Glu Met 850 855 860 Asp Glu GluArg Glu Met Leu Val Asp Glu Glu Tyr Glu Leu Tyr Gln 865 870 875 880 AspPro Ser Pro Ser Met Glu Leu Tyr Pro Leu Ser His Ile Gln Glu 885 890 895Ala Thr Pro Val Pro Ser Val Asn Glu Leu His Phe Gly Thr Gln Trp 900 905910 Leu His Asp Asn Glu Pro Ser Glu Ser Gln Glu Ala Arg Thr Gly Arg 915920 925 Thr Val Tyr Ser Gln Glu Ala Gln Pro Tyr Gly Tyr Cys Pro Glu Asn930 935 940 Val Met Lys Glu Asn Phe Val Met Glu Ser Leu Pro Ser Val ProSer 945 950 955 960 Thr Glu Gly Asn Ser Gln Gln Gly Arg Phe Asp Asp LeuGlu Asn Leu 965 970 975 Asn Ser Leu Ala Lys Thr Ser Leu Asp Leu Gly MetIle Pro Asn Asp 980 985 990 Val Gln Gly Pro Ser Leu Leu Ile Asp Leu ProVal Val Ala Gln Arg 995 1000 1005 Arg Glu Gln Glu Asp Leu Pro Leu TyrGln His Gln Ala Thr Arg Val 1010 1015 1020 Ile Ser Lys Ala Ser Ala TyrThr Gly Met Leu Ser Ser Arg Tyr Ala 1025 1030 1035 1040 Thr Asp Thr CysGlu Leu Pro Glu Arg Glu Glu Gly Glu Gly Glu Glu 1045 1050 1055 Thr ProAsn Phe Ser His Trp Gly Pro Pro Arg Ile Val Glu Ile Phe 1060 1065 1070Arg Glu Pro Asn Val Ser Leu Gly Ile Ser Ile Val Gly Gly Gln Thr 10751080 1085 Val Ile Lys Arg Leu Lys Asn Gly Glu Glu Leu Lys Gly Ile PheIle 1090 1095 1100 Lys Gln Val Leu Glu Asp Ser Pro Ala Gly Lys Thr AsnAla Leu Lys 1105 1110 1115 1120 Thr Gly Asp Lys Ile Leu Glu Val Ser GlyVal Asp Leu Gln Asn Ala 1125 1130 1135 Ser His Ser Glu Ala Val Glu AlaIle Lys Asn Ala Gly Asn Pro Val 1140 1145 1150 Val Phe Ile Val Gln SerLeu Ser Ser Thr Pro Arg Val Ile Pro Asn 1155 1160 1165 Val His Asn LysAla Asn Lys Ile Thr Ser Asn Gln Asn Gln Asp Thr 1170 1175 1180 Gln GluLys Lys Glu Lys Arg Gln Gly Thr Ala Pro Pro Pro Met Lys 1185 1190 11951200 Leu Pro Pro Pro Tyr Lys Ala Leu Thr Asp Asp Ser Asp Glu Asn Glu1205 1210 1215 Glu Glu Asp Ala Phe Thr Asp Gln Lys Ile Arg Gln Arg TyrAla Asp 1220 1225 1230 Leu Pro Gly Glu Leu His Ile Ile Glu Leu Glu LysAsp Lys Asn Gly 1235 1240 1245 Leu Gly Leu Ser Leu Ala Gly Asn Lys AspArg Ser Arg Met Ser Ile 1250 1255 1260 Phe Val Val Gly Ile Asn Pro GluGly Pro Ala Ala Ala Asp Gly Arg 1265 1270 1275 1280 Met His Ile Gly AspGlu Leu Leu Glu Ile Asn Asn Gln Ile Leu Tyr 1285 1290 1295 Gly Arg SerHis Gln Asn Ala Ser Ala Ile Ile Lys Thr Ala Pro Ser 1300 1305 1310 LysVal Lys Leu Val Phe Ile Arg Asn Glu Asp Ala Val Asn Gln Met 1315 13201325 Ala Val Thr Pro Phe Pro Val Pro Ser Ser Ser Pro Ser Ser Ile Glu1330 1335 1340 Asp Gln Ser Gly Thr Glu Pro Ile Ser Ser Glu Glu Asp GlySer Leu 1345 1350 1355 1360 Glu Val Gly Ile Lys Gln Leu Pro Glu Ser GluSer Phe Lys Leu Ala 1365 1370 1375 Val Ser Gln Met Lys Gln Gln Lys TyrPro Thr Lys Val Ser Phe Ser 1380 1385 1390 Ser Gln Glu Ile Pro Leu AlaPro Ala Ser Ser Tyr His Ser Thr Asp 1395 1400 1405 Ala Asp Phe Thr GlyTyr Gly Gly Phe Gln Ala Pro Leu Ser Val Asp 1410 1415 1420 Pro Ala ThrCys Pro Ile Val Pro Gly Gln Glu Met Ile Ile Glu Ile 1425 1430 1435 1440Ser Lys Gly Arg Ser Gly Leu Gly Leu Ser Ile Val Gly Gly Lys Asp 14451450 1455 Thr Pro Leu Asn Ala Ile Val Ile His Glu Val Tyr Glu Glu GlyAla 1460 1465 1470 Ala Ala Arg Asp Gly Arg Leu Trp Ala Gly Asp Gln IleLeu Glu Val 1475 1480 1485 Asn Gly Val Asp Leu Arg Asn Ser Ser His GluGlu Ala Ile Thr Ala 1490 1495 1500 Leu Arg Gln Thr Pro Gln Lys Val ArgLeu Val Val Tyr Arg Asp Glu 1505 1510 1515 1520 Ala His Tyr Arg Asp GluGlu Asn Leu Glu Ile Phe Pro Val Asp Leu 1525 1530 1535 Gln Lys Lys AlaGly Arg Gly Leu Gly Leu Ser Ile Val Gly Lys Arg 1540 1545 1550 Asn GlySer Gly Val Phe Ile Ser Asp Ile Val Lys Gly Gly Ala Ala 1555 1560 1565Asp Leu Asp Gly Arg Leu Ile Gln Gly Asp Gln Ile Leu Ser Val Asn 15701575 1580 Gly Glu Asp Met Arg Asn Ala Ser Gln Glu Thr Val Ala Thr IleLeu 1585 1590 1595 1600 Lys Cys Ala Gln Gly Leu Val Gln Leu Glu Ile GlyArg Leu Arg Ala 1605 1610 1615 Gly Ser Trp Thr Ser Ala Arg Thr Thr SerGln Asn Ser Gln Gly Ser 1620 1625 1630 Gln Gln Ser Ala His Ser Ser CysHis Pro Ser Phe Ala Pro Val Ile 1635 1640 1645 Thr Gly Leu Gln Asn LeuVal Gly Thr Lys Arg Val Ser Asp Pro Ser 1650 1655 1660 Gln Lys Asn SerGly Thr Asp Met Glu Pro Arg Thr Val Glu Ile Asn 1665 1670 1675 1680 ArgGlu Leu Ser Asp Ala Leu Gly Ile Ser Ile Ala Gly Gly Arg Gly 1685 16901695 Ser Pro Leu Gly Asp Ile Pro Val Phe Ile Ala Met Ile Gln Ala Ser1700 1705 1710 Gly Val Ala Ala Arg Thr Gln Lys Leu Lys Val Gly Asp ArgIle Val 1715 1720 1725 Ser Ile Asn Gly Gln Pro Leu Asp Gly Leu Ser HisAla Asp Val Val 1730 1735 1740 Asn Leu Leu Lys Asn Ala Tyr Gly Arg IleIle Leu Gln Val Val Ala 1745 1750 1755 1760 Asp Thr Asn Ile Ser Ala IleAla Ala Gln Leu Glu Asn Met Ser Thr 1765 1770 1775 Gly Tyr His Leu GlySer Pro Thr Ala Glu His His Pro Glu Asp Thr 1780 1785 1790 Glu Thr ProPro Pro Lys Ile Ile Thr Leu Glu Lys Gly Ser Glu Gly 1795 1800 1805 LeuGly Phe Ser Ile Val Gly Gly Tyr Gly Ser Pro His Gly Asp Leu 1810 18151820 Pro Ile Tyr Val Lys Thr Val Phe Ala Lys Gly Ala Ala Ala Asp Asp1825 1830 1835 1840 Gly Arg Leu Lys Arg Gly Asp Gln Ile Leu Ala Val AsnGly Glu Thr 1845 1850 1855 Leu Glu Gly Val Thr His Glu Gln Ala Val AlaIle Leu Lys His Gln 1860 1865 1870 Arg Gly Thr Val Thr Leu Thr Val Leu1875 1880 4 290 DNA Mus musculus CDS (105)..(290) 4 acttccgccaggtgaggagg ccgtccgtgc ccgcagcccc ggggctccca ccccgccgtc 60 gcccgatcagactttttgga agtgattgaa aagaatatcc caaa atg cct gaa aac 116 Met Pro GluAsn 1 cct gct gca gag aag atg cag gtc ctg cag gtc ctg gat cgc ctt cga164 Pro Ala Ala Glu Lys Met Gln Val Leu Gln Val Leu Asp Arg Leu Arg 5 1015 20 ggg aag ctg cag gag aag gga gac acg acg cag aac gag aag ctg tct212 Gly Lys Leu Gln Glu Lys Gly Asp Thr Thr Gln Asn Glu Lys Leu Ser 2530 35 gcg ttc tac gag acg ctg aag agc cct ctc ttc aac cag atc ctt aca260 Ala Phe Tyr Glu Thr Leu Lys Ser Pro Leu Phe Asn Gln Ile Leu Thr 4045 50 ctg cag cag tcc atc aag cag ctg aag gga 290 Leu Gln Gln Ser IleLys Gln Leu Lys Gly 55 60 5 62 PRT Mus musculus 5 Met Pro Glu Asn ProAla Ala Glu Lys Met Gln Val Leu Gln Val Leu 1 5 10 15 Asp Arg Leu ArgGly Lys Leu Gln Glu Lys Gly Asp Thr Thr Gln Asn 20 25 30 Glu Lys Leu SerAla Phe Tyr Glu Thr Leu Lys Ser Pro Leu Phe Asn 35 40 45 Gln Ile Leu ThrLeu Gln Gln Ser Ile Lys Gln Leu Lys Gly 50 55 60 6 15 PRT Homo sapiens 6Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile Thr Lys Val 1 5 10 15 716 PRT Artificial Sequence Description of Artificial SequenceSH3 BindingProtein PDZ Domain 7 Ser Gly Ser Gly Ile Leu Ala Pro Pro Val Pro Pro ArgAsn Thr Arg 1 5 10 15 8 16 PRT Artificial Sequence Description ofArtificial SequenceAF6 PDZ Binding Protein 8 Ser Gly Asp Asp Gly Asp AspPro Phe Leu Gln Tyr Glu Phe Tyr Val 1 5 10 15 9 16 PRT Homo sapiens 9Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile Thr Lys Val 1 5 1015 10 20 DNA Artificial Sequence Description of Artificial SequenceMMSC1Primers 10 caggtgaggc agggccgaca 20 11 22 DNA Artificial SequenceDescription of Artificial SequenceMMSC1 Primers 11 ctacagtagg cagggcaacagg 22 12 38 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 12 gttttcccag tcacgacgcg ggctcccacc tgctcctc 38 1341 DNA Artificial Sequence Description of Artificial SequenceMMSC1Primers 13 aggaaacagc tatgaccatg tgaacactaa caaacctttc c 41 14 40 DNAArtificial Sequence Description of Artificial SequenceMMSC1 Primers 14gttttcccag tcacgacgtc aactcaacca tataccctca 40 15 40 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 15 aggaaacagctatgaccatg gctggacatc cttcacgaag 40 16 38 DNA Artificial SequenceDescription of Artificial SequenceMMSC1 Primers 16 gttttcccag tcacgacggccttggattca gtgtggtg 38 17 40 DNA Artificial Sequence Description ofArtificial SequenceMMSC1 Primers 17 aggaaacagc tatgaccatc cccaacaaactgtttcaggc 40 18 38 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 18 gttttcccag tcacgacgcc agggaaccag tccacaca 38 1938 DNA Artificial Sequence Description of Artificial SequenceMMSC1Primers 19 aggaaacagc tatgaccatc ctgactgaat tcccacag 38 20 23 DNAArtificial Sequence Description of Artificial SequenceMMSC1 Primers 20tcctggagga ttagcagatc gag 23 21 24 DNA Artificial Sequence Descriptionof Artificial SequenceMMSC1 Primers 21 ggtaatccaa aatgctgaat ccca 24 2239 DNA Artificial Sequence Description of Artificial SequenceMMSC1Primers 22 gttttcccag tcacgacgaa gattggtggc acaaacgtg 39 23 41 DNAArtificial Sequence Description of Artificial SequenceMMSC1 Primers 23aggaaacagc tatgaccata gcactgccag gtattatact t 41 24 41 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 24 gttttcccagtcacgacgag aattgttggc tatgttggaa c 41 25 41 DNA Artificial SequenceDescription of Artificial SequenceMMSC1 Primers 25 aggaaacagc tatgaccatgctccagttag aaagagagct g 41 26 39 DNA Artificial Sequence Description ofArtificial SequenceMMSC1 Primers 26 gttttcccag tcacgacgac atcctcatctacttctcca 39 27 40 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 27 aggaaacagc tatgaccata actcagcatc atctgcaatc 4028 38 DNA Artificial Sequence Description of Artificial SequenceMMSC1Primers 28 gttttcccag tcacgacggg aaaacctgtt gggtcctg 38 29 42 DNAArtificial Sequence Description of Artificial SequenceMMSC1 Primers 29aggaaacagc tatgaccatc gacagcaaac caaagtaaaa gg 42 30 22 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 30 gtggattcctttgatgggca cc 22 31 23 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 31 ctttgagcca caacaggaag gtc 23 32 39 DNAArtificial Sequence Description of Artificial SequenceMMSC1 Primers 32gttttcccag tcacgacgtg agctgcttga ggtcaatgg 39 33 39 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 33 aggaaacagctatgaccatc taaagggtcc tggtaatcc 39 34 39 DNA Artificial SequenceDescription of Artificial SequenceMMSC1 Primers 34 gttttcccag tcacgacgcccctgaagtca agattgttg 39 35 44 DNA Artificial Sequence Description ofArtificial SequenceMMSC1 Primers 35 aggaaacagc tatgaccata caactttcttcttcattatc ttcc 44 36 38 DNA Artificial Sequence Description ofArtificial SequenceMMSC1 Primers 36 gttttcccag tcacgacgga aatattgaaagctgtgcc 38 37 40 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 37 aggaaacagc tatgaccatg tcagaaattc atgcatctcc 4038 40 DNA Artificial Sequence Description of Artificial SequenceMMSC1Primers 38 gttttcccag tcacgacgaa agtctttcca ttcccaacaa 40 39 39 DNAArtificial Sequence Description of Artificial SequenceMMSC1 Primers 39aggaaacagc tatgaccatc catacggctg tgcctcctg 39 40 25 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 40 gagttatatcaagatccctc accat 25 41 23 DNA Artificial Sequence Description ofArtificial SequenceMMSC1 Primers 41 caaatatgct catgcgtgat cgg 23 42 41DNA Artificial Sequence Description of Artificial SequenceMMSC1 Primers42 gttttcccag tcacgacgtt cactttggta cacagtggtt g 41 43 40 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 43 aggaaacagctatgaccata aatcttcttg ctccctcctt 40 44 39 DNA Artificial SequenceDescription of Artificial SequenceMMSC1 Primers 44 gttttcccag tcacgacgcccgaatgatgt ccaaggtcc 39 45 40 DNA Artificial Sequence Description ofArtificial SequenceMMSC1 Primers 45 aggaaacagc tatgaccatg tccaccaacaatactgatcc 40 46 38 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 46 gttttcccag tcacgacgag ccactggggt ccaccgag 38 4741 DNA Artificial Sequence Description of Artificial SequenceMMSC1Primers 47 aggaaacagc tatgaccata ctcgtggagt ggatgacaaa c 41 48 38 DNAArtificial Sequence Description of Artificial SequenceMMSC1 Primers 48gttttcccag tcacgacgca gttgaggcca ttaagaat 38 49 42 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 49 aggaaacagctatgaccatc aagttcaata atgtgcagtt ct 42 50 23 DNA Artificial SequenceDescription of Artificial SequenceMMSC1 Primers 50 cgccaatgaa acttcctcctcct 23 51 23 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 51 tctcctgtga ggcatttctc atg 23 52 41 DNAArtificial Sequence Description of Artificial SequenceMMSC1 Primers 52gttttcccag tcacgacgcc tttaccgacc aaaaaatcag a 41 53 38 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 53 aggaaacagctatgaccatc tgattgactg catcctcg 38 54 40 DNA Artificial SequenceDescription of Artificial SequenceMMSC1 Primers 54 gttttcccag tcacgacgcatctgccatta ttaagactgc 40 55 41 DNA Artificial Sequence Description ofArtificial SequenceMMSC1 Primers 55 aggaaacagc tatgaccatg tgaagtctgcatctgttgaa t 41 56 40 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 56 gttttcccag tcacgacgtc caacaaaagt ctccttcagt 4057 40 DNA Artificial Sequence Description of Artificial SequenceMMSC1Primers 57 aggaaacagc tatgaccata acctctaata tctggtcacc 40 58 39 DNAArtificial Sequence Description of Artificial SequenceMMSC1 Primers 58gttttcccag tcacgacgct atagttatcc atgaagtct 39 59 38 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 59 aggaaacagctatgaccatc cgcctttcac gatgtcag 38 60 22 DNA Artificial SequenceDescription of Artificial SequenceMMSC1 Primers 60 gaaggtgcgg ctggtggtgtat 22 61 21 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 61 cttgctctgt cacccaggct g 21 62 38 DNA ArtificialSequence Description of Artificial SequenceMMSC1 Primers 62 gttttcccagtcacgacggg cctgagcatc gttgggaa 38 63 40 DNA Artificial SequenceDescription of Artificial SequenceMMSC1 Primers 63 aggaaacagc tatgaccataaccaggtttt gcaggccagt 40 64 39 DNA Artificial Sequence Description ofArtificial SequenceMMSC1 Primers 64 gttttcccag tcacgacgtc agggtagtcagcagagtgc 39 65 39 DNA Artificial Sequence Description of ArtificialSequenceMMSC1 Primers 65 aggaaacagc tatgaccatt acccacatcc gcgtgagac 39

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (a) a human MMSC1 polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 3; (b) a mutated human MMSC1polypeptide obtainable by expression of a mutated form of the nucleicacid set forth in SEQ ID NO: 2; and (c) a mutant human MMSC1 polypeptidewhich cannot form a complex with a wild-type protein with whichwild-type MMSC1 does form a complex.
 2. The isolated polypeptide ofclaim 1 which is a human MMSC1 polypeptide comprising the amino acidsequence set forth in SEQ ID NO:
 3. 3. The isolated polypeptide of claim1 which is a mutated human MMSC1polypeptide obtainable by expression ofa mutated form of the nucleic acid set forth in SEQ ID NO:
 2. 4. Theisolated polypeptide of claim 1 which is an isolated mutant human MMSC1polypeptide which cannot form a complex with a wild-type protein withwhich wild-type MMSC1 does form a complex.
 5. The isolated polypeptideof claim 4, wherein said wild-type protein is MMAC1.
 6. The isolatedpolypeptide of claim 1 which is labeled.
 7. The isolated polypeptide ofclaim 1 in the form of a fusion protein.
 8. An isolated protein complexselected from the group consisting of: (a) a protein complex comprisingMMSC1 and MMAC1 and (b) a protein complex comprising a fragment of MMSC1and a fragment of MMAC1.
 9. The isolated protein complex of claim 8which is protein complex comprising MMSC1 and MMAC1.
 10. The isolatedprotein complex of claim 9, wherein said MMSC1 contains an alteration.11. The isolated protein complex of claim 8, wherein said MMAC1 containsan alteration.
 12. The isolated protein complex of claim 8 which is acomplex of a fragment of MMSC1 and a fragment of MMAC1.
 13. The proteincomplex of claim 12, wherein said fragment of MMSC1 comprises PDZ domainnumber
 7. 14. The protein complex of claim 12, wherein said MMSC1comprises an alteration.
 15. The protein complex of claim 12, whereinsaid MMAC1 comprises an alteration.
 16. The protein complex of claim 13,wherein said MMSC1 comprises an alteration.
 17. The protein complex ofclaim 13, wherein said MMAC1 comprises an alteration.
 18. A method fordetecting an alteration in MMSC1 wherein said alteration is associatedwith cancer in a human, wherein if said alteration is in germline it isassociated with predisposition to said cancer and if said alteration isin somatic tissue it indicates that said somatic tissue is cancerous,wherein said method comprises analyzing a MMSC1 gene expression productfrom a tissue of said human.
 19. The method of claim 18, wherein saidexpression product is selected from the group consisting of a MMSC1polypeptide encoded by the MMSC1 gene.
 20. The method of claim 19wherein one or more of the following procedures is carried out: (a)immunoblotting; (b) immunocytochemistry; (c) assaying for bindinginteractions between MMSC1 protein isolated from said tissue and abinding partner capable of specifically binding the polypeptideexpression product of a MMSC1 mutant allele and/or a binding partner forthe MMSC1 polypeptide having the amino acid sequence set forth in SEQ IDNO: 3; and (d) assaying for the inhibition of biochemical activity ofsaid binding partner.
 21. The method of claim 20 wherein said alterationof MMSC1 protein is detected by assaying for binding interactionsbetween said MMSC1 protein isolated from said tissue and MMAC1 protein.22. A method for detecting an alteration in MMAC1 wherein saidalteration is associated with cancer in a human, wherein if saidalteration is in germline it is associated with predisposition to saidcancer and if said alteration is in somatic tissue it indicates thatsaid somatic tissue is cancerous, wherein said method comprisesanalyzing an MMAC1 polypeptide from a tissue of said human by assayingfor binding interactions between said MMAC1 polypeptide and MMSC1 or PDZdomain number 7 of said MMSC1.
 23. A method for supplying a wild-typeMMSC1 gene function or a MMSC1 function substantially similar towild-type to a cell which has lost said gene function or has alteredgene function by virtue of a mutation in said MMSC1 gene, wherein saidmethod comprises introducing into said cell a molecule which suppressesa transformed state of said cell, said molecule selected from the groupconsisting of all or a part of a wild-type MMSC1 polypeptide which isrequired for non-neoplastic growth of said cell, a polypeptidesubstanially homologous to said wild-type MMSC1 polypeptide and amolecule which mimics the function of said wild-type MMSC1 polypeptide.24. A method for diagnosing a predisposition for cancer in a humanwherein said method comprises assaying for the ability of MMSC1 or afragment of MMSC1 from said human to form a complex with a protein towhich wild-type MMSC1 binds wherein an inability to form said complex isindicative of a predisposition to cancer.
 25. The method of claim 24,wherein said protein is MMAC1.
 26. The method of claim 24, wherein saidassay comprises measuring in vitro a complex formed by mixing saidprotein and MMSC1 purified from said human.
 27. The method of claim 24,wherein said assay comprises measuring in vitro a complex formed bymixing MMSC1 and said protein purified from said human.
 28. The methodof claim 24, wherein said complex is measured by binding with anantibody specific for a MMSC1-said protein complex.
 29. The method ofclaim 24, wherein said assay comprises mixing an antibody specific for aMMSC1-said protein complex with a tissue extract from said person,wherein the lack of formation of a MMSC1-said protein-antibody complexbetween said antibody and said tissue extract is indicative of apredisposition to cancer.
 30. A method for determining whether amutation in a protein to which MMSC1 binds is predispositive for cancerwherein said method comprises binding said protein with said mutation toa wild-type MMSC1 and determining whether a complex forms, wherein thelack of a complex indicates said mutation is predispositive.
 31. Amethod for determining whether a mutation in MMSC1 is predispositive forcancer wherein said method comprises binding a MMSC1 with said mutationto a protein to which wild-type MMSC1 binds and determining whether acomplex forms, wherein the lack of a complex indicates said mutation ispredispositive.